Robert Kruklitis

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.

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.

Transbronchial Needle Injection: A Systematic Review of a New Diagnostic and Therapeutic Paradigm

Background and Objective: Transbronchial needle catheters are commonly used during flexible and rigid bronchoscopy for needle aspiration. The use of these catheters can be expanded by employing the technique of transbronchial needle injection. Methods and Results: By injecting lesions in the airways, peribronchial structures, mediastinum, or lung parenchyma, transbronchial needle injection has been applied to the treatment of lung cancer, inflammatory disorders of the airways, recurrent respiratory papillomatosis, as well as bronchopleural fistulas. Diagnostic applications have included the localization of peripheral lung nodules as well as sentinel lymph nodes. Conclusions: Our review defines this bronchoscopic technique and summarizes its various reported applications.

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.

Stent-Mediated Gene Delivery for Site-Specific Transgene Administration to the Airway Epithelium and Management of Tracheobronchial Tumors

Background: Gene therapy is currently under investigation as a means of managing a variety of pulmonary diseases. Unfortunately, gene transfer to bronchial epithelium has been hampered by the lack of stable and efficient transduction. Recent studies have shown that gene vectors could be tethered to the metallic surfaces of intra-arterial stents. This approach enables efficacious and site-specific adenoviral gene delivery to the vascular endothelium. Objectives: We hypothesized that airway mesh stents impregnated with viral gene vectors could be used for local gene delivery to benign and malignant bronchial epithelium. Methods: Serotype 5 adenoviral vectors (Ad5, E1-/E3-) containing the reporter genes green fluorescent protein (Ad.GFP) or β-galactoside/LacZ (Ad.LacZ), or a therapeutic gene, Ad.INF-β, were coupled to either metallic mesh disks or stents via anti-Ad knob antibodies. These platforms were assessed for their ability to transfect bronchial epithelial cells from both rats and humans, as well as murine (L1C2) and human (A549) lung cancer cell lines. Gene transfer was quantified by fluorescent microscopy, scanning fluorimetry for Ad.GFP, and light microscopy studies assessing β-galactosidase staining for Ad.LacZ. Metallic mesh and stent-mediated gene transfer was also performed in a murine flank tumor model and in a rat endotracheal tumor model in order to evaluate the therapeutic potential. Results: In these studies, murine and human non-small cell lung cancer (NSCLC) cells were successfully transfected with reporter genes in vitro. Ad.LacZ-complexed mesh successfully transfected reporter genes into established murine flank NSCLC tumors. In addition, Ad.LacZ-tethered stents could effectively transfect both tracheobronchial epithelium and submucosal glands in rats. Similar epithelial transfection was achieved in ex vivo human bronchial epithelium. Pilot in vivo experimentation provided data supporting the concept that therapeutic genes could also be delivered with this technology. In additional pilot in vivo experiments, the growth of murine flank tumors was inhibited by placement of mesh disks coupled with Ad.muINF-β, and rats bearing endotracheal tumors demonstrated a trend towards prolonged survival with insertion of Ad.ratINF-β-tethered stents. Conclusions: Stent-mediated gene delivery successfully enabled site-specific vector administration to target rat and human airway cells in cell culture, organ culture and in vivo. Local tracheobronchial gene delivery via stents could provide a viable clinical solution for overcoming the difficulties encountered with vector delivery within the lungs, in particular by lowering requisite vector titers and by directing desired vectors to areas of interest. This strategy may prove valuable for treating tumors involving the tracheobronchial tree, as well as other nonmalignant tracheobronchial disorders.