Analysis of eIF4E and 4EBP1 mRNAs in head and neck cancer.

OBJECTIVES/HYPOTHESIS: The eukaryotic translation initiation factor 4E (eIF4E) in conjunction with its binding protein, 4EBP1, regulates the translation of cap-dependent mRNAs. An aberrant increase in eIF4E shifts the balance in favor of translation of transcripts that promote cell proliferation and malignancy. eIF4E protein is commonly elevated in head and neck squamous cell carcinomas (HNSCC), and its overexpression is associated with increased recurrence. An underlying mechanism for eIF4E overexpression is gene amplification, and we wanted to determine whether eIF4E mRNA could serve as a prognostic maker of HNSCC. METHODS: Tumor specimens from 26 HNSCC patients and oral tissues from 17 control subjects were examined for eIF4E and 4EBP1 by semiquantitative RT-PCR and correlated with clinical and pathologic findings. RESULTS: Unlike eIF4E mRNA alone, expression of eIF4E relative to 4EBP1 was a more precise predictor of HNSCC and its progression (P < .01, Wilcoxon rank sum test). Eight of 26 patients (31%) had elevated eIF4E:4EBP1 (4E:4EBP1; >25), and 7 of these (87.5%) had recurrence. Alternately, from 18 patients with low 4E:4EBP1 (<25; 69%), only 5 patients had recurrence (30.1%). To determine the probability of no recurrence, Kaplan-Meier analysis showed significantly poor disease-free survival in patients with elevated 4E:4EBP1 than those with low ratios (P < .01, log rank test). CONCLUSIONS: Elevated 4E:4EBP1 significantly correlated with increased disease recurrence. Because 4EBP1 modulates eIF4E activity, our results highlight the importance of incorporating a joint analysis of eIF4E and 4EBP1 mRNAs in HNSCC patient care decisions.

Human papillomavirus in metastatic lymph nodes from unknown primary head and neck squamous cell carcinoma.

OBJECTIVE: Determine human papillomavirus (HPV) incidence in unknown primary squamous cell carcinomas (SCCa) of the head and neck and assess if HPV status influenced survival. STUDY DESIGN: Historical cohort study. SETTING: Tertiary care center. SUBJECTS: Patients with unknown primary SCCa despite a complete workup who underwent neck dissection or excisional biopsy and postoperative comprehensive ± chemoradiotherapy between 2002 and 2009. METHODS: HPV fluorescence in situ hybridization (FISH) and p16(INK4a) immunohistochemistry (p16 IHC) were performed. Results were compared with survival, age, race, gender, tobacco use, alcohol use, and nodal stage. RESULTS: Twenty-five patients met the inclusion criteria, of whom 88% were >10 pack year tobacco users. Twenty-eight percent were HPV-positive defined by both p16+ and FISH+. Five-year overall survival was 66.7% in HPV-positive and 48.5% in HPV-negative patients (P = .35). Similarly, 5-year disease-free survival rates were 66.7% in HPV-positive and 48.5% in HPV-negative patients (P = .54). All 3 HPV-positive nonsmokers were survivors, but this was not significant because of the small sample size (P > .05). No other characteristics were associated with survival (P > .05). CONCLUSION: Twenty-eight percent of metastatic lymph nodes from occult primary tumors were HPV positive. There was no survival difference associated with HPV status. Most of the HPV-positive patients in this study were tobacco users who had similar survival to HPV-negative patients, so caution should be used in interpreting HPV status in these patients.

Curcumin inhibits carcinogen and nicotine-induced Mammalian target of rapamycin pathway activation in head and neck squamous cell carcinoma.

Curcumin appears to be a safe, bioactive food compound that is a potential chemopreventive for patients at a high risk for head and neck squamous cell carcinoma (HNSCC). Identification and validation of intermediate endpoints is an important step in evaluating chemopreventive agents. AKT/MTOR pathway biomarkers are intrinsic to the carcinogenic process as well as the mechanism of intervention with curcumin. Antiproliferative effects of curcumin were assayed in 9 HNSCC and a keratinocyte cell line. Nicotine, a genotoxic alkaloid involved in tobacco addiction, forms DNA adducts and has been implicated in upper aerodigestive tract cancer promotion. The antiproliferative effects of curcumin were associated with inhibition of the AKT/MTOR pathway in presence and absence of nicotine, which also induced this pathway. Curcumin was highly effective at suppressing growth of SCC40 xenografts and its activity is associated with modulation of MTOR's downstream target pS6. Curcumin at 15 mg significantly increased survival (286 ± 37 vs. 350 days) in the 4NQO carcinogenic model survival study. A major cause of lethal progression of HNSCC is local regional migration and invasion of malignant cells, and curcumin significantly inhibited cancer cell migration and invasion in vitro and in vivo where downregulation of pS6 was associated with a significant decrease in MMP-9. This is the first study to demonstrate that curcumin inhibits the adverse effects of nicotine by blocking nicotine-induced activation of the AKT/MTOR pathway in HNSCC, which retards cell migration. These studies indicate that inhibiting the AKT/MTOR pathway with curcumin may be useful as an oral chemopreventive agent.

Molecular analysis of surgical margins in head and neck squamous cell carcinoma patients.

OBJECTIVES/HYPOTHESIS: Molecular analysis of surgical margins is playing an increasingly important role in establishing surgical margins. Most markers lack the sensitivity and ease of applicability for effective clinical use. To date, the proto-oncogene eIF4E (4E) is elevated in 100% of head and neck squamous cell carcinoma tumors and is of prognostic value in predicting recurrence. In a retrospective study, 4E overexpression in the margins appeared to be a more sensitive predictor of recurrence when compared with p53. The goal was to confirm this finding in a prospective study and also to compare the expression of matrix metalloproteinase-9 (MMP-9) to 4E expression in tumors and margins. Other objectives were to determine which of these markers have prognostic significance in predicting recurrence and elucidate whether there is any additional benefit to analysis of surgical margins with a combination of the three molecular markers. STUDY DESIGN: A prospective study was performed on all patients who consecutively underwent primary surgical resection between 1998 and 1999 for head and neck squamous cell carcinoma. Patient and tumor characteristics were reviewed, and time to recurrence was noted. METHODS: Paraffin-embedded sections of tumors and all histologically tumor-free margins were analyzed for the presence or absence of 4E, p53, and MMP-9 with immunohistochemical analysis. Patients were followed according to the institution's head and neck cancer protocol, and time to recurrence was noted. RESULTS: Ninety-eight percent of tumors overexpressed 4E, 65% overexpressed p53, and 92% overexpressed MMP-9. Of the 52 patients with tumor-free margins, 52%, 46%, and 54% had positive margins for 4E, p53, and MMP-9, respectively. Although no significant correlation between 4E and p53 expression was seen in the margins (P =.16), a significant correlation between 4E and MMP-9 expression was noted (P =.0002). However, when expression of 4E and p53 in the margins of only the patients who overexpressed p53 in the tumors was compared (n = 34), there was a significant correlation (P =.04). There was also a significant difference in the disease-free interval between patients with 4E-positive and 4E-negative margins (P =.003). This difference in time to recurrence was not significant for the p53-positive versus the p53-negative group (P =.18) but approached significance when MMP-9 was used as a marker (P =.07). Although the univariate analysis showed that stage, nodal disease, grade, and 4E expression in the margins were significantly associated with disease-free interval, in the Cox multiple regression analysis, only 4E expression in the margin was significantly associated with disease-free interval (P =.01). CONCLUSIONS: The era for molecular analysis of surgical margins is here. Although a significant correlation was seen between 4E and MMP-9, overexpression of 4E appears to be a significant predictor of recurrence when compared with the well-studied tumor suppressor gene p53 and a relatively novel marker, MMP-9.