Mammalian target of rapamycin inhibitors as possible adjuvant therapy for microscopic residual disease in head and neck squamous cell cancer.

Molecular therapeutics identifies an aberration in tumors to select patients that benefit from molecular targeted therapy. Overexpression of eIF4E in histologically "tumor-free" surgical margins of head and neck squamous cell cancer (HNSCC) patients is an independent predictor of recurrence and is functionally activated through the Akt/mammalian target of rapamycin (mTOR) pathway. Although mTOR inhibitors are cytostatic agents, best used in combination therapy, we hypothesize that they can be used as long-term single agents in an HNSCC model of minimal residual disease (MRD). CCI-779, an mTOR inhibitor, arrested growth of a phosphatase and tensin homologue deleted on chromosome 10 (PTEN) abnormal HNSCC cell line FaDu, inhibiting phosphorylation of 4E-binding protein 1, resulting in increased association with eIF4E and inhibition of basic fibroblast growth factor and vascular endothelial growth factor. Fluorescence in situ hybridization detected PTEN abnormalities in 68% of patient tumors and 35% of tumor-free margins. CCI-779 inhibited growth of established tumors in nude mice. However, in the MRD model, there were significant differences in the tumor-free rate between the control (4%) and the treatment group (50%), and the median tumor-free time was 7 versus 18 days, respectively (P < 0.0001). In those animals that formed tumors, CCI-779 caused a significant decrease in the tumor volume. The Kaplan-Meier curve showed that CCI-779 significantly increased survival (P < 0.0001). The mTOR pathway was inhibited in peripheral blood mononuclear cells potential surrogate markers of response to therapy. Stable transfection of FaDu with luciferase allowed us to monitor the effects of CCI-779 with bioluminescence imaging in the MRD model. These results pave the way for a clinical trial using targeted molecular therapy with CCI-779 as a single agent for mTOR-activated residual cells.

Overexpressed eIF4E is functionally active in surgical margins of head and neck cancer patients via activation of the Akt/mammalian target of rapamycin pathway.

PURPOSE: Overexpression of eIF4E in surgical margins of head and neck cancer patients is an independent risk factor for recurrence. We hypothesize that overexpressed eIF4E is functionally active in tumor margins through activation of the Akt/mammalian target of rapamycin (mTOR) pathway EXPERIMENTAL DESIGN: Western blots and/or immunohistochemistry were performed to determine whether phosphorylation of mTOR and activation of its downstream molecules eIF4E-binding protein-1 (4E-BP1) and p70 S6 kinase and the upstream modulator of mTOR, Akt, were expressed in margins overexpressing eIF4E. RESULTS: There was a significant association between phospho-4E-BP1 and eIF4E expression of a margin or a significant difference in phospho-4E-BP1 expression between the eIF4E-positive and -negative margins (P < 0.01). A significant association between eIF4E and phospho-p70 S6 kinase as well as eIF4E and phospho-mTOR was also noted (P < 0.05). Western blot analysis indicated a highly significant difference in the phosphorylation status of 4E-BP1 between tumors and resection margins. A total of 89% of the 4E-BP1-expressing margins expressed more of the phosphorylated (beta, gamma, and delta) isoforms, whereas 81% of the 4E-BP1-expressing tumors expressed more of the unphosphorylated alpha isoform. A similar difference in Akt activation was noted between eIF4E-positive margins and tumors (P < 0.05). CONCLUSIONS: Overexpression of eIF4E is functionally active in tumor margins through activation of the Akt/mTOR signaling pathway. The greater degree of expression of downstream targets and upstream regulators of mTOR in margins compared with the tumors indicates preferential activation of the Akt/mTOR signaling pathway in margins overexpressing eIF4E. Rapamycin analogs can potentially be used as adjuvant therapy for patients with eIF4E-positive margins.

Predictive factors for posterior triangle metastasis in HNSCC.

OBJECTIVE: Surgical modifications sparing uninvolved structures such as the spinal accessory nerve have been implemented since the advent of the radical neck dissection in 1906. The increased morbidity to the spinal accessory nerve involved with the dissection of level V lymph nodes has led to much controversy. In this study, we examine the incidence of nodal metastasis to all nodal levels involved with upper aerodigestive squamous cell carcinoma and attempt to determine when level V dissection is indicated. STUDY DESIGN: Retrospective chart review. METHODS: A study of all radical and modified radical neck dissections was performed at Louisiana State University - Shreveport Health Sciences Center and Overton Brooks Veterans Administration Hospital between 1996 and 2003 for upper aerodigestive squamous cell carcinoma. Univariate and multivariate analyses were performed to determine which neck and patient factors were significantly associated with level V metastasis. RESULTS: Seventy-nine patients with a total of 94 neck dissections were analyzed. The prevalence of level V metastasis was 7.4% of the total neck dissections. Multivariate analysis found that positive lymph nodes involving levels II, III, and IV was the only independent significant factor for level V metastasis (P = .0003). CONCLUSION: Our study is in concordance with other studies in the literature, revealing a low prevalence of level V metastasis in upper aerodigestive squamous cell carcinoma. Unlike other studies, we have found if levels II, III, and IV lymph nodes are found to be positive, dissection of level V is warranted.

NS 398 radiosensitizes an HNSCC cell line by possibly inhibiting radiation-induced expression of COX-2.

PURPOSE: Cyclooxygenase-2 (COX-2) protein is frequently elevated in squamous cell carcinoma of the head and neck (HNSCC). The aim of this study was to determine if COX-2 inhibitors have radiosensitizing effects in HNSCC and understand the mechanism by which this occurs. MATERIALS AND METHODS: The radiosensitizing effects of a selective COX-2 inhibitor, NS398, on a HNSCC cell line HEp3, were determined using clonogenic survival assay. Cells were pretreated with the dose of NS398 at which 50% growth inhibition occurred (IC(50)) and then irradiated. COX-2 protein and mRNA were then determined in the presence and absence of NS398. RESULTS: NS398 significantly decreased (p < 0.0001) the calculated survival fraction (SF) for all radiation doses (0.79 to 0.41 at 2 Gy). A significant increase in COX-2 protein of 2.8 fold for 2 Gy and 3.5 fold for 6 Gy was noted 48 h after radiation. Interestingly, the upregulation of COX-2 protein with radiation was suppressed when cells were pretreated with NS398. Quantitative reverse transcriptase polymerase chain reaction showed no significant corresponding increase in COX-2 mRNA at 48 h with ionizing radiation. CONCLUSIONS: The radiosensitizing effect of NS398 could be due to inhibition of radiation-induced COX-2 upregulation by this drug. NS398, known as an inhibitor of COX-2 enzyme activity, down-regulated COX-2 protein expression, which may indicate that NS398 can act upstream of COX-2, and this change appears to be post-transcriptional.

Pharmacodynamic evaluation of temsirolimus in patients with newly diagnosed advanced-stage head and neck squamous cell carcinoma.

BACKGROUND: Activation of the mammalian target of rapamycin (mTOR) pathway in surgical margins of head and neck squamous cell carcinoma (HNSCC) is a predictor of recurrence and patients with minimal residual disease may benefit from adjuvant therapy with temsirolimus, an mTOR inhibitor. METHODS: The effects of 3 weekly doses of 25 mg of temsirolimus on Akt/mTOR pathway biomarkers were evaluated in tumor and peripheral blood mononuclear cells (PBMCs) of patients with HNSCC. Adverse events were assessed. RESULTS: Temsirolimus significantly decreased pS6 and p4E-BP1 in tumors, and pS6 and pAkt in PBMCs (p < .05). There was no significant upregulation of pAkt(Ser(473)) in tumor tissue. Side effects were minimal and reversible. CONCLUSION: Significant inhibition of the mTOR pathway was noted in both tumors and PBMCs of HNSCC with minimal side effects. The mTOR inhibitors can potentially be used as adjuvant therapy for patients with minimal residual disease and PBMCs are potential surrogate markers in this setting.

Modulation of the cell growth regulator mTOR by Epstein-Barr virus-encoded LMP2A.

Control of translation initiation is one means by which cells regulate growth and proliferation, with components of the protein-synthesizing machinery having oncogenic potential. Expression of latency protein LMP2A by the human tumor virus Epstein-Barr virus (EBV) activates phosphatidylinositol 3-kinase/Akt located upstream of an essential mediator of growth signals, mTOR (mammalian target of rapamycin). We show that mTOR is activated by expression of LMP2A in carcinoma cells, leading to wortmannin- and rapamycin-sensitive inhibition of the negative regulator of translation, eukaryotic initiation factor 4E-binding protein 1, and increased c-Myc protein translation. Intervention by this DNA tumor virus in cellular translational controls is likely to be an integral component of EBV tumorigenesis.