Peter DeLong

Long-term Follow-up of Patients with Malignant Pleural Mesothelioma Receiving High-Dose Adenovirus Herpes Simplex Thymidine Kinase/Ganciclovir Suicide Gene Therapy

Purpose: Delineation of the long-term follow-up data on a series of patients with malignant mesothelioma, who received a single intrapleural dose of a nonreplicative adenoviral (Ad) vector encoding the herpes simplex virus thymidine kinase “suicide gene” (Ad.HSVtk) in combination with systemic ganciclovir. Experimental Design: This report focuses on the 21 patients receiving “high-dose” therapy, defined by an intrapleural dose of vector (≥1.6 × 1013 viral particles), where transgene-encoded tk protein was reliably identified on immunohistochemical staining. In 13 patients, the vector was deleted in the E1 and E3 regions of the Ad; in the other eight patients, the vector had deletions in the Ad genes E1 and E4. Safety, immunologic responses, transgene expression, and clinical responses were evaluated. Results: Both the E1/E3-deleted vector and the E1/E4-deleted vector were well tolerated and safe, although production of the E1/E4 vector was more difficult. Posttreatment antibody responses against the tumors were consistently seen. Interestingly, we observed a number of clinical responses in our patients, including two long-term (>6.5 year) survivors, both of whom were treated with the E1/E4-deleted vector. Conclusions: Intrapleural Ad.HSVtk/ganciclovir is safe and well tolerated in mesothelioma patients and resulted in long-term durable responses in two patients. Given the limited amount of gene transfer observed, we postulate that Ad.HSVtk may have been effective due to induction of antitumor immune responses. We hypothesize that approaches aiming to augment the immune effects of Ad gene transfer (i.e., with the use of cytokines) may lead to increased numbers of therapeutic responses in otherwise untreatable pleural malignancies.

Regulatory T cells and cytokines in malignant pleural effusions secondary to mesothelioma and carcinoma

Immunotherapy against a variety of malignancies, including pleural-based malignancies, has shown promise in animal models and early human clinical trials, but successful efforts will need to address immuno-suppressive factors of the tumor and host, particularly certain cytokines and CD4+ CD25+ regulatory T cells (Treg). Here, we evaluated the cellular and cytokine components of malignant pleural effusions from 44 patients with previously diagnosed mesothelioma, non-small cell lung cancer (NSCLC), or breast cancer and found significant differences in the immune profile of pleural effusions secondary to mesothelioma vs. carcinoma. Although a high prevalence of functionally suppressive CD4+ CD25+ T cells was found in carcinomatous pleural effusions, mesothelioma pleural effusions contained significantly fewer CD4+ CD25+ T cells. Activated CD8+ T cells in pleural fluid were significantly more prevalent in mesothelioma than carcinoma. However, there is clear patient-to-patient variability and occasional mesothelioma patients with high percentages of CD4+ CD25+ pleural effusion T cells and low percentages of CD8+ CD25+ pleural effusion T cells can be identified. Mesothelioma pleural effusions contained the highest concentrations of the immuno-suppressive cytokine transforming growth factcor (TGF)-beta. Thus, the contribution of cellular and cytokine components of immuno-suppression associated with malignant pleural effusions varies by tumor histology and by the individual patient. These results have implications for the development of immunotherapy directed to the malignant pleural space, and suggest the need to tailor immunotherapy to overcome immunosuppressive mechanisms in tumor environments.

Tumor necrosis factor α modulates airway smooth muscle function via the autocrine action of interferon β

Current evidence suggests that tumor necrosis factor α (TNFα) and the family of interferons (IFNs) synergistically regulate many cellular responses that are believed to be critical in chronic inflammatory diseases, although the underlying mechanisms of such interaction are complex, cell-specific, and not completely understood. In this study, TNFα in a time-dependent manner activated both janus tyrosine kinase 1 and Tyk2 tyrosine kinase and increased the nuclear translocation of interferon-regulatory factor-1, STAT1, and STAT2 in human airway smooth muscle cells. In cells transfected with a luciferase reporter, TNFα stimulated γ-activated site-dependent gene transcription in a time- and concentration-dependent manner. Using neutralizing antibodies to IFNβ and TNFα receptor 1, we show that TNFα-induced secretion of IFNβ mediated γ-activated site-dependent gene expression via activation of TNFα receptor 1. In addition, neutralizing antibody to IFNβ also completely abrogated the activation of interferon stimulation response element-dependent gene transcription induced by TNFα. Secreted IFNβ acted as a negative regulator of TNFα-induced interleukin-6 expression, while IFNβ augmented TNFα-induced RANTES (regulated on activation normal T cell expressed and secreted) secretion but had little effect on TNFα-induced intercellular adhesion molecule-1 expression. Furthermore TNFα, a modest airway smooth muscle mitogen, markedly induced DNA synthesis when cells were treated with neutralizing anti-IFNβ. Together these data show that TNFα, via the autocrine action of IFNβ, differentially regulates the expression of proinflammatory genes and DNA synthesis.

Alterations in cell cycle genes in early stage lung adenocarcinoma identified by expression profiling.

In normal lung epithelial cells, cellular division is an ordered, tightly regulated process involving multiple checkpoints that assess extracellular growth signals, cell size, and DNA integrity. In contrast, neoplastic lung cells develop the ability to bypass several of these checkpoints, particularly at the G1/S and G2/M boundaries. We used genomic profiling to compare gene expression levels in early stage lung adenocarcinomas and non-neoplastic pulmonary tissue in order to comprehensively identify alterations in the process of cell cycling. RNA extracted from node negative, poorly differentiated lung adenocarcinomas (15 patients) and non-neoplastic pulmonary tissue (5 patients) was hybridized to oligonucleotide microarray filters containing 44,363 genes. Ontological classification was used to extract genes involved with cell cycle progression. Further analysis discovered a subset of differentially expressed genes for further study. Of the 624 cell cycle genes on the microarray filters, 40 genes were predicted to be differentially expressed in lung adenocarcinomas. Alterations in several genes (ie. cyclin B1, cyclin D1, p21, MDM2) are consistent with published data in the literature. We also identified 19 novel genes that have neither been described in non-small cell lung cancer (ie. cdc2, cullin 4A, ZAC, p57, DP-1, GADD45, PISSLRE, cdc20) nor in any other tumors (ie. cyclin F, cullin 5, p34). These results identified several potential cell cycle genes altered in lung cancer.

Soluble Type II Transforming Growth Factor-β Receptor Inhibits Established Murine Malignant Mesothelioma Tumor Growth by Augmenting Host Antitumor Immunity

Purpose: Transforming growth factor (TGF)-β blockade has been proposed as an anticancer therapy; however, understanding which tumor patients might benefit most from such therapy is crucial. An ideal target of such inhibitory therapy might be malignant mesothelioma (MM), a highly lethal, treatment-resistant malignancy of mesothelial cells of the pleura and peritoneum that produces large amounts of TGF-β. The purpose of this study was to explore the possible therapeutic utility of TGF-β blockade on MM. Experimental Design: To evaluate this hypothesis, we tested the effects of a soluble TGF-β type II receptor (sTGF-βR) that specifically inhibits TGF-β1 and TGF-β3 in three different murine MM tumor models, AB12 and AC29 (which produce large amounts of TGF-β) and AB1 (which does not produce TGF-β). Results: Tumor growth of both established AB12 and AC29 tumors was inhibited by sTGF-βR. In contrast, AB1 tumors showed little response to sTGF-βR. The mechanism of these antitumor effects was evaluated and determined to be primarily dependent on immune-mediated responses because (a) the antitumor effects were markedly diminished in severe combined immunodeficient mice or mice depleted of CD8+ T cells and (b) CD8+ T cells isolated from spleens of mice treated with sTGF-βR showed strong antitumor cytolytic effects, whereas CD8+ T cells isolated from spleens of tumor-bearing mice treated with of control IgG2a showed no antitumor cytolytic effects. Conclusions: Our data suggest that TGF-β blockade of established TGF-β-secreting MM should be explored as a promising strategy to treat patients with MM and other tumors that produce TGF-β.

Treatment of lung cancer using clinically relevant oral doses of the cyclooxygenase-2 inhibitor rofecoxib: potential value as adjuvant therapy after surgery.

Objective:To investigate the uses and limitations of cyclooxygenase- (COX) 2 inhibition using clinically relevant doses of oral rofecoxib in the treatment of murine models of non–small-cell lung cancer (NSCLC). Summary Background Data:Overexpression of COX-2 has been reported in lung cancer. Several studies have demonstrated that high doses of COX-2 inhibitors could inhibit the growth of rodent and human lung cancer cell lines. The potential uses and limitations of COX-2 inhibition at doses equivalent to those currently approved for use in humans have not been well studied. Methods:Three murine NSCLC cell lines were injected into the flanks of mice to establish tumor xenografts. Mice were treated orally with low doses of a COX-2 inhibitor (rofecoxib chow, 0.0075%). Mechanisms were evaluated by analysis of tumor-infiltrating lymphocytes. To study rofecoxib as adjuvant therapy, large established tumors (14–18 days after tumor inoculation) were surgically debulked and animals were treated with rofecoxib starting 3 days before surgery. Recurrence of the tumor after debulking was monitored. Results:Rofecoxib significantly slowed the growth of small (0-120 mm3) tumors (P < 0.01-0.05) in all 3 cell lines, with higher efficacy in the more immunogenic tumors. Minimal responses were noted in larger tumors. Rofecoxib appeared to augment CD8+ T cell infiltration in immunogenic tumors. Rofecoxib significantly reduced the recurrence rate after debulking (P < 0.01). Conclusions:Clinically relevant doses of the COX-2 inhibitor rofecoxib given orally were effective in inhibiting the growth of small (but not large) tumors in 3 murine NSCLC cell lines tested and in preventing recurrences after surgical debulking. Depending on the immunogenicity of human tumors, COX-2 inhibition might be useful as adjuvant therapy for surgically resectable NSCLC.

Differentially Expressed Apoptotic Genes in Early Stage Lung Adenocarcinoma Predicted by Expression Profiling

OBJECTIVE: In undiseased lung epithelial cells, apoptosis is an evolutionarily conserved and genetically regulated form of cell suicide which plays an important role in development and in the maintenance of tissue homeostasis. Neoplastic lung cells develop the ability to deregulate growth by alterations in these genes which control apoptosis. Genomic profiling was used to compare gene expression levels in early stage lung adenocarcinomas and non-neoplastic pulmonary tissue in order to comprehensively identify alterations in the process of apoptosis. METHODS: RNA extracted from node negative, poorly differentiated lung adenocarcinomas (15 patients) and non-neoplastic pulmonary tissue (5 patients) was hybridized to oligonucleotide microarray filters containing 44,363 genes. Ontological classification was used to extract genes involved with apoptosis. Further analysis discovered a subset of differentially expressed genes for further study. RESULTS: Of the 308 apoptotic genes on the microarray filters, 24 genes were predicted to be differentially expressed in lung adenocarcinomas. Alterations in several genes (ie. Akt, BcL-xL, PTEN, FAS) are consistent with the literature. We also identified 10 novel genes that have not been described in non-small cell lung cancer (ie. RIP, Caspase 1, PDK-1). CONCLUSIONS: These results identified several potential apoptotic genes altered in lung cancer.