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Despite focused research in conventional therapies, the 5-year survival rate of lung cancer patients remains only 15%. Due to the lack of reliable early diagnostic techniques, most patients are diagnosed with advanced-stage disease and therefore have a poor prognosis. There are more than 100 million current and former smokers in the US who have elevated risk for lung cancer and may benefit from a non-invasive test for early detection. Patients at risk may have subclinical disease for years prior to diagnosis. Therefore the clinical challenge is to develop lung cancer detection methods that are effective during this window of opportunity to increase the rate of early detection and thereby spur early treatment and improve lung cancer patient outcomes. During tumorigenesis, the developing tumor forms a complex cellular microenvironment that produces many soluble mediators, such as proteins and miRNAs, which are important for tumor initiation and growth and thus can serve as lung cancer biomarkers. Because the pulmonary vascular bed comprises approximately 50% of the body’s total vasculature, the peripheral blood is expected to reflect even small variations in lung cancer biomarkers.

Over the past decade, our laboratory has gathered substantial data supporting the analysis of soluble mediators of lung carcinogenesis including inflammatory cytokines, growth and angiogenic factors, and miRNA. Additionally, a considerable literature has developed describing the contribution of these factors to lung carcinogenesis. Based on these findings, in our preliminary studies we have developed a plasma protein-based biomarker panel that accurately distinguished between lung cancer patients and at-risk control subjects. Our predictive model provided 97% sensitivity, 77% specificity, and an area under the ROC of .93 for stage 1 vs. high-risk controls. Because recent studies demonstrated that circulating miRNA can serve as robust and specific diagnostic biomarkers, we developed a panel of 17 miRNAs relevant to lung carcinogenesis. In our preliminary studies utilizing this miRNA panel, we were able to detect miRNAs that are differentially expressed in the plasma of lung cancer patients as compared to high-risk controls. We hypothesize that the proteins and miRNA profiles will each contribute unique information and thus, an integrated panel of protein and miRNA markers may provide higher sensitivity for early lung cancer detection than either platform alone.

Specific aims for the current proposal include: 1) to identify protein profiles in plasma associated with early-stage lung cancer, 2) to identify plasma miRNA profiles associated with early stage lung cancer, and 3) to integrate miRNA and cytokine biomarkers established in Aims 1 and 2 to improve early-stage lung cancer detection. The first and second aims address the clinical problems of patients at risk for lung cancer who present with a suspicious pulmonary nodule of indeterminate significance that has be further evaluated.

Lung cancer is the leading cause of death for men and women in the United States. Screening methods for the early detection of lung cancer must be developed to circumvent the phenomenon that sees the majority of patients being diagnosed with advanced, and thus incurable, lung cancer. While computerized tomography was demonstrated to reduce mortality by 20% in a high-risk smoker patient cohort, further examination of this method, complementary screening methods, and tools to screen less at-risk populations are needed to make the detection of early-stage lung cancer most beneficial. Using the expertise of our multidisciplinary clinical lung cancer team (Drs. Kelly, Gandara, Cooke, Yoneda, and Murin) with leaders in metabolomics and lipid biology (Dr. Fiehn) and glycomics (Drs. Miyamoto and Lebrilla), we aim to identify mass spectroscopy-detectible biomarkers of early lung cancer. The advent of “omics” technologies will allow us to expand the current pool of DNA and protein biomarkers and facilitate our vision of a systems-biology screening and/or diagnostic tool based on “biomarker panels.” Our approach begins with a discovery phase that will identify biomarkers in tumor and blood with the aim of finding blood-based biomarkers that most closely reflect tumor biology. Next we will proceed to the testing phase to determine the performance of these biomarkers in four independent cohorts (two lung cancer cohorts, healthy controls, and benign nodules). Biomarkers that meet our pre-specified criteria will require further evaluation that is beyond the scope of this proposal.

Cancer-associated fibroblasts (CAFs) support the growth of lung cancer cells in vivo by secretion of soluble factors that stimulate the growth of tumor cells. One such soluble factor is CLCF1. Functional studies in our laboratory identified an important role for CLCF1–CNTFR signaling in promoting growth of NSCLCs. NSCLC cell lines as well as patient-derived xenografts were found to express CNTFR, and knock-down of CNTFR leads to decreased proliferation. To test the use of CNTFR blockade as an anti-cancer therapy, we developed a soluble version of CNTFR (i.e., a CNTFR “receptor decoy”) that can be expressed from an adenoviral vector (Ad-sCNTFR-Fc). Preliminary experiments indicate that Ad-sCNTFR-Fc can block proliferation of a NSCLC cell line in vitro. Our proposed studies build on an established translational research program that includes intensive efforts to harvest and analyze primary tumor samples and CAFs directly from patients. In Aim 1, we will test the therapeutic efficacy of Ad-sCNTFR-Fc in a panel of patient-derived xenografts (PDX) as well as established cell lines. In Aim 2, to increase therapeutic efficacy, we will use protein engineering to develop a high-affinity sCNTFR that can more effectively block CLCF1-CNTFR interactions, and will evaluate this therapeutic candidate in PDX models. In Aim 3, we will establish the utility of using plasma CLCF1 as a biomarker for CLCF1 production in CAFs isolated directly from patient tumors. Parallel analysis of CAF and plasma CLCF1 will establish if CLCF1 in plasma correlates with CLCF1 in tumors and is thus a useful biomarker.

Small cell lung carcinoma (SCLC) remains one of the most lethal human cancers. More than 25,000 patients are diagnosed with SCLC every year in the United States. Unfortunately, most patients usually survive only 6-12 months after diagnosis. Except for the combination of radiation therapy and chemotherapy, there has been no novel therapeutic approach implemented in the past 30 years. Despite a large number of clinical trials, no targeted therapy has been approved for SCLC patients, whose survival rate hovers around 5%. Previous attempts to identify therapeutic targets in SCLC patients have mostly focused on signaling networks within the tumor cells. Here we propose to test a novel immunotherapeutic approach to promote the anti-cancer activity of macrophages. Specifically, we found that SCLC cells express high levels of the CD47 antigen, a marker of self that inhibits the phagocytic activity of macrophages. Based on previous studies from the laboratories of Dr. Weissman and Dr. Garcia at Stanford, we propose to block the interaction between CD47 and SIRPa, an inhibitory receptor expressed on macrophages, to stimulate the engulfment of SCLC cells by macrophages and their eradication. Furthermore, we will identify SCLC-specific antigens and combine CD47-blocking therapies with these antibodies to enhance the recruitment and the activity of macrophages against SCLC cells. Clinical trials testing the CD47-blocking strategies are being initiated in patients with blood cancer and some solid tumors. If successful, our pre-clinical experiments may lead to the rapid implementation of this novel anti-cancer immunotherapy strategy in SCLC patients.