A link between Rel B expression and tumor progression in laryngeal cancer

Abstract

Laryngeal cancer is a frequent malignancy originating from the squamous vocal epithelium in a multi-stage fashion in response to environmental carcinogens. Although most cases can be cured by surgery and/or radiotherapy, advanced and relapsing disease is common, and biomarkers of such dismal cases are urgently needed. The cancer genome of laryngeal cancers was recently shown to feature a signature of aberrant nuclear factor (NF)-κB activation, but this finding has not been clinically exploited. We analyzed primary tumor samples of 96 well-documented and longitudinally followed patients covering the whole spectrum of laryngeal neoplasia, including 21 patients with benign laryngeal diseases, 15 patients with dysplasia, 43 patients with early-stage carcinoma, and 17 patients with locally advanced carcinoma, for immunoreactivity of RelA, RelB, P50, and P52/P100, the main NF-κB subunits that activate transcription. Results were cross-examined with indices of tumor progression and survival. Interestingly, RelB expression increased with tumor stage, grade, and local extent. Moreover, patients displaying high RelB immunoreactivity exhibited statistically significantly poorer survival compared with patients featuring low levels of RelB expression (P = 0.018 by log-rank test). Using Cox regression analyses and tumor stage, local extent, grade and RelA/RelB immunoreactivity, we develop a new score that can independently predict survival of patients with laryngeal cancer. Hence we provide a simple and affordable NF-κB-based test to predict prognosis in laryngeal cancer.

Keywords: biomarker; head and neck squamous cell carcinoma; immunohistochemistry; nuclear factor (NF)-κB; prognosis.

Figure 1. Study design and survival of 96 patients with benign and malignant laryngeal disease

(A) Schematic representation of patient clinicopathologic categories and their distribution across the spectrum of laryngeal neoplasia. (B) Overall Kaplan-Meier survival plot with 95% confidence interval. (C-I) Kaplan-Meier survival estimates of patients stratified by gender (C; female: n = 10; male: n = 86), age (D; ≤65 years: n=49; >65 years: n=47), smoking (E; ≤100 pack years: n= 37; >100 pack years: n= 59), alcohol (F; no: n= 48; yes: n= 48), clinicopathologic category (G; benign/dysplasia: n = 36; carcinoma: n = 60), TNM7 stage (H; I: n = 55; II-IV: n = 41), and tumor grade (I; none/low: n = 47; medium/high: n = 49). n, sample size; P, probability by log-rank test.

Figure 2. Immunohistochemical detection of NF-κB subunit expression by clinicopathologic study group

(A) Data summary shown as raw data points and bars (median) with boxes (interquartile range) and whiskers (95% percentiles). (B) Representative images.n, sample size; P, overall probability by Kruskal-Wallis test. ^* and ^**: P< 0.05 and P< 0.01, respectively, for comparison with benign group by Dunn’s post- tests. ^#: P< 0.05 for comparison with dysplasia group by Dunn’s post-tests.

Figure 3. NF-κB subunit expression by TNM7 stage

(A) Data summary shown as raw data points and bars (median) with boxes (interquartile range) and whiskers (95% percentiles). (B) Representative images.n, sample size; P, overall probability by Kruskal-Wallis test. ^**: P< 0.01 for comparison with benign group by Dunn’s post-tests.

Figure 4. Immunohistochemical detection of NF-κB subunit expression by tumor grade

Data summary shown as raw data points and bars (median) with boxes (interquartile range) and whiskers (95% percentiles). n = 39, 8, 31, and 18, respectively, for none (not applicable or specified), low, intermediate, and high grade groups. P, overall probability by Kruskal-Wallis test. ^*: P< 0.05 for comparison with benign group by Dunn’s post-tests.

Figure 5. Survival by NF-κB subunit expression

Shown are Kaplan-Meier survival estimates of patients dichotomized by median NF-κB subunit expression score (n = 48/group for all groups and graphs). n, sample size; P, probability by log-rank test.

Figure 6. Cox regression analysis of the impact of clinical variables, risk factors, and NF-κB subunit expression on survival

(A) Risk ratios (RR) with 95% confidence intervals (CI) and probability values (P) of the of the independent impact of the listed variables on survival. Note that successive variable only emerged as important after elimination of the preceeding variables. Equation showing the proposed laryngeal cancer prognostic score. (B) Frequency distribution of study cohort according to the newly devised score showing the two groupings with low (0-25; n = 70) and high (> 25; n = 26) scores. (C) Kaplan-Meier survival estimates of laryngeal cancer patients with low (0-25) and high (> 25) scores. P, probability by log-rank test. (D) Cox regression survival estimates of laryngeal cancer patients with low (0-25) and high (> 25) scores. P, probability by proportional hazards model. RR, Risk ratio of high versus low score. CI, 95% confidence interval.

Figure 7

Receiver-operator curve (ROC) analysis of the impact of clinical variables, risk factors, and NF-κB subunit expression on tumor histology (A), stage (B), and progression (C). AUC, area under curve; P, probability.