Background. Cellular immune suppression is observed in head and neck squamous cell cancer (HNSCC) and contributes to poor prognosis. Restoration of immune homeostasis may require primary cell-derived cytokines at physiologic doses. An immunotherapy regimen containing a biologic, with multiple-active cytokine components, and administered with cytoxan, zinc, and indomethacin was developed to modulate cellular immunity.Methods. Study methods were designed to determine the safety and efficacy of a 21-day neoadjuvant immunotherapy regimen in a phase 2 trial that enrolled 27 therapy-naive patients with stage II to IVa HNSCC. Methods included safety, clinical and radiologic tumor response, disease-free survival (DFS), overall survival (OS), and tumor lymphocytic infiltrate (LI) data collection.Results. Acute toxicity was minimal. Patients completed neoadjuvant treatment without surgical delay. By independent radiographic review, 83% had stable disease during treatment. OS was 92%, 73%, and 69% at 12, 24, and 36 months, respectively. Histologic analysis suggested correlation between survival and tumor LI.Conclusion. Immunotherapy regimen was tolerated. Survival results are encouraging. (C) 2011 Wiley Periodicals, Inc. Head Neck 33: 1666-1674, 2011

Abstract The Wnt signaling pathway is critical to normal development but the involvement of the pathway in lung tumorigenesis has not been clearly established. Wnt7a, with its receptor Fzd9, is required for maintenance of epithelial differentiation and inhibits transformed growth. Wnt7a has been shown to be frequently lost in NSCLC cell lines and tumors. We demonstrate that loss of Wnt7a in a non-transformed lung epithelial cell line leads to increased proliferation. Using pyrosequencing, 58 of 81 (72%) of NSCLC tumors we tested had higher methylation in the Wnt7a promoter region compared to adjacent normal lung tissue. We also tested 10 pairs of human NSCLC tumor and normal adjacent lung samples with known loss of expression of Wnt7a in the tumor and 6 (60%) had increased methylation of the Wnt7a promoter region in the tumors. Two NSCLC cell lines demonstrated to have methylated Wnt7a and loss of Wnt7a expression, H157 and H1299, were used to investigate methylation of the Wnt7a promoter. Treatment of these cell lines with 5-aza-2deoxycytidine resulted in increased expression of Wnt7a and increased downstream activity of known targets of Wnt7a-Fzd9 signaling. Methylation of a Wnt7a promoter luciferase resulted in decreased activity of the luciferase compared to an untreated Wnt7a luciferase. NNK is a tobacco carcinogen known to attenuate DNMT1 degradation, leading to increased promoter methylation. Treatment of a non-transformed lung epithelial cell line with NNK resulted in decreased expression of Wnt7a and increased clonogenicity. Together, these results indicate that Wnt7a is frequently lost by methylation in NSCLC and that methylation of Wnt7a leads to decreased expression. Wnt7a could be methylated by exposure to tobacco carcinogens, leading to loss of expression and increased transformed lung cell growth. The clinical use of demethylating agents could restore Wnt7a activity, helping to decrease growth of NSCLC. Citation Format: {Authors}. {Abstract title} [abstract]. In: Proceedings of the 102nd Annual Meeting of the American Association for Cancer Research; 2011 Apr 2-6; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2011;71(8 Suppl):Abstract nr 88. doi:10.1158/1538-7445.AM2011-88

Objectives:Head and neck squamous cell carcinoma (HNSCC) is associated with profound defects in cellular immunity. IRX-2, a primary cell-derived biologic containing multiple cytokines, has enhanced immune responses and induced tumor rejection in preclinical studies. This phase 1 open label study aimed to determine the clinical and laboratory safety of an IRX-2 regimen in patients with HNSCC. Methods:Patients with HNSCC who had failed surgery and/or radiation therapy were enrolled. IRX-2 was injected subcutaneously at 115 units per dose, 2 doses/d over 10 days, starting on day 4. Patients received low-dose cyclophosphamide infusion on day 1 and took oral indomethacin and zinc daily from day 1 through day 21. Safety and laboratory assessments were undertaken throughout the treatment and 4 weeks after completion of the regimen. Results:A total of 13 patients with advanced disease were enrolled in the safety/intent-to-treat population; all experienced treatment-emergent adverse events (AEs). The most frequent AEs were blood and lymphatic disorders, followed by gastrointestinal disorders. Most AEs were mild to moderate in severity. Three patients discontinued the study due to an AE, including 2 deaths. Two patients died after the study period due to tumor progression. No death or discontinuation was considered related to the study drugs. Antitumor responses were noted by radiographic assessment. In the 8 patients who had antitumor data at day 21, 1 patient had complete response, 5 had stable disease, and 2 had progressive disease. Conclusions:The IRX-2 regimen was tolerated in patients with advanced HNSCC who failed surgery and/or radiation therapy. The safety and antitumor activity observed warrants further studies.

Sprouty proteins are potent receptor tyrosine kinase inhibitors that antagonize growth factor signaling and are involved in lung development. However, little is known about the regulation or targets of Sprouty-4 (Spry4) in lung cancer. Our study aimed to determine the role of Spry4 in non–small cell lung cancer (NSCLC). We found that Spry4 mRNA expression was decreased in NSCLC cell lines and in dysplastic lung cell lines compared with a nontransformed cell line, suggesting that Spry4 has tumor-suppressing activity. When Spry4 was stably transfected into H157 and H2122 NSCLC cell lines, decreased migration and invasion were observed. Matrix metalloproteinase-9 activity was decreased, and the expression of matrix metalloproteinase inhibitors TIMP1 and CD82 were increased. Stable expression of Spry4 led to reduced cell growth and reduced anchorage-independent growth in NSCLC cell lines, along with upregulation of tumor suppressors p53 and p21. Changes in epithelial and mesenchymal markers indicated that Spry4 expression induces a reversal of the epithelial to mesenchymal transition characteristic of tumor cells. Treatment of a nontransformed lung epithelial cell line with short hairpin RNA to Spry4 led to the decreased expression of epithelial markers and increased cell growth, supporting the concept of Spry4 acting as a tumor suppressor. We showed that the activity of the Spry4 promoter is increased by Wnt7A/Fzd9 signaling through peroxisome proliferator–activated receptor γ. These data present previously undescribed targets of Spry4 and suggest that Spry4 is a downstream target of Wnt7A/Fzd 9 signaling. Spry4 may have efficacy in the treatment of NSCLC. Mol Cancer Res; 8(6); 833–43. ©2010 AACR.

The goal of this study was to assess the ability of iloprost, an orally active prostacyclin analog, to inhibit transformed growth of human non-small cell lung cancer (NSCLC) and to define the mechanism of iloprost's tumor suppressive effects. In a panel of NSCLC cell lines, the ability of iloprost to inhibit transformed cell growth was not correlated with the expression of the cell surface receptor for prostacyclin, but instead was correlated with the presence of Frizzled 9 (Fzd 9) and the activation of peroxisome proliferator-activated receptor-γ (PPARγ). Silencing of Fzd 9 blocked PPARγ activation by iloprost, and expression of Fzd 9 in cells lacking the protein resulted in iloprost's activation of PPARγ and inhibition of transformed growth. Interestingly, soluble Frizzled-related protein-1, a well-known inhibitor of Wnt/Fzd signaling, also blocked the effects of iloprost and Fzd 9. Moreover, mice treated with iloprost had reduced lung tumors and increased Fzd 9 expression. These studies define a novel paradigm, linking the eicosanoid pathway and Wnt signaling. In addition, these data also suggest that prostacyclin analogs may represent a new class of therapeutic agents in the treatment of NSCLC where the restoration of noncanonical Wnt signaling maybe important for the inhibition of transformed cell growth.