Non-small cell lung cancer (NSCLC)

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.

The overarching concept guiding our work is that combinations of biomarkers and imaging characteristics will be most robust in predicting which patients with lung nodules should receive an aggressive diagnostic and/or therapeutic approach and which patients should be monitored for nodule stability over time. Our objectives are to improve two biomarker tests that already have shown significant promise-- sputum chromosomal aneusomy by fluorescence in situ hybridization (CA-FISH) and the analysis of multiple serum analytes by a highly multiplexed aptamer-based assay developed by SomaLogic, Inc.-- and then to compare test performance with predictive models based on nodule and patient characteristics. These objectives are based on two separate hypotheses: 1. The current CA-FISH assay panel can be further improved by the development of two sub-panels, one for detection of squamous cell carcinoma of the lung (SCC) and the second for the detection of adenocarcinoma of the lung (AC). 2. The current aptamer-based analysis can be further improved by developing separate assays specifically designed for the detection of AC, SCC, K-ras, and EGFR-mutated lung tumors. The assays will then be applied to sputum and blood specimens from the Pittsburgh SPORE Pittsburgh Lung Screening Study (PLuSS) and Colorado SPORE Lung Nodule cohorts, in concert with analysis of the CT characteristics of the corresponding lung nodules, including size, volume, density, location, or airway involvement, to compare test performances and to assess whether combining tests would be valuable.

Evaluating suspicious pulmonary nodules in the setting of a lung cancer diagnostic work-up is a common clinical management challenge. High resolution multi-detector computed tomography visualizes many noncalcified lesions that could represent either potentially lethal cancers or benign processes. Although the vast majority of these nodules are benign, non-neoplastic lesions, a subset will be malignant and in these cases, patients could benefit from immediate treatment. A non-invasive test that maintains a high sensitivity and specificity across the spectrum of clinical presentations of pulmonary nodules would be of tremendous clinical benefit by reducing the number unnecessary invasive procedures and minimizing the time between detection and definitive treatment. The overall goal for the proposed studies is to develop a convenient and low-cost serum test that will, either alone or in combination with clinical features, accurately classify indeterminate nodules and direct proper clinical management. Our approach to develop such a test will be to profile and identify which proteins detected in the serum at the time of the detection of these nodules were immunogenic and induced the production of circulating autoantibodies. Differences in the autoantibody profile between the malignant and non-malignant groups will then be further evaluated against additional clinical specimens from both our institution and two pre-specified collaborator institutions to identify the optimal test for this urgent clinical need. Once completed, we anticipate this test will aid clinicians in deciding the optimal treatment for patients found to have suspicious nodules and improve the current standard of care.

Melanoma has the highest propensity to metastasize to the brain once the disease has spread, while non-small cell lung cancer (NSCLC) has the highest incidence of brain metastases among all cancers. Approximately 50,000 patients develop brain metastases from these diseases every year. Patients with brain metastases have exceedingly limited therapeutic options since they are typically excluded from clinical trials. Recently a number of new systemic immune therapies, such as nivolumab (BMS-936558) and lambrolizumab (MK-3475), two inhibitors of PD-1 (an immune inhibitory molecule), have led to dramatic responses in metastatic melanoma (MM) and NSCLC. However, these therapies have not been studied in patients with untreated brain metastases.  Little is known about immune cells in the brain, and whether the immune system can be stimulated to reject cancer cells. Moreover, little is known about what tumor characteristics are associated with response to MK-3475 in brain metastases. This proposal includes innovative correlative biomarker studies incorporated into a clinical trial uniquely designed to study the activity of MK-3475 in patients with limited, small brain metastases in MM and NSCLC. This drug carries the hope to provide prolonged responses similar to responses seen in other metastatic sites, and might enable patients to avoid radiation therapy, and improve their outcome. As part of this clinical trial, we will collect brain and other metastatic specimens from patients enrolled and study the expression of immune inhibitory molecules utilizing a novel quantitative assay and algorithms that we have developed in preliminary work. These studies will facilitate selection of patients who are more likely to respond to this class of drugs.

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.

Advanced (stage IV) lung adenocarcinoma is an incurable disease. Treatment mostly consists of chemotherapy, which provides a consistent but small survival benefit. Standard therapy includes a platinum-doublet (pemetrexed or paclitaxel) often in combination with bevacizumab, followed by maintenance bevacizumab or pemetrexed. Programmed death 1 (PD-1) is a T-cell surface receptor that when activated, delivers a negative signal to T cells. This feedback mechanism dampens immune responses when no longer needed, preventing damaging autoimmune reactions. Tumors can co-opt this T-cell control mechanism to escape surveillance and/or rejection by the immune system. Thus, reversing this tumor action can result in antitumor activity worthy of clinical evaluation. Recently it has been demonstrated that inhibition of the PD-1 receptor with an anti-PD-1 monoclonal antibody (nivolumab) has significant clinical activity in lung cancer with an ORR of 26%. As remarkable as this may be, given the heavily pretreated population of lung cancer patients studied, this also indicates that 74% of patients did not respond and remained resistant to this approach. Therefore, going forward it is important to develop combination therapies to improve the efficacy of this approach, and to discover resistance mechanisms that may allow for better patient (and tumor) selection. One potential class of agents to be used in combination with anti-PD-1 is the adenosine A2AR antagonists. Given that A2AR on T cells within the tumor microenvironment contributes to immune escape and therapeutic resistance, the addition of PBF-509 is expected to improve the efficacy of nivolumab.

Lung cancer remains the leading cause of cancer-related death due to the limited ability to detect the disease at an early and potentially curable stage. Low-dose computerized tomography (LDCT) screening of high-risk patients improves mortality. However, the high proportion of false positive tests, the majority of which are pulmonary nodules, has limited widespread adoption of this life-saving screening modality. Since screen-detected pulmonary nodules contribute to the overall cost of screening and potential morbidity due to the need for further diagnostic testing, there is an urgent need for accurate tools to stratify patients with screen-detected nodules for routine follow-up or additional intervention. Our data suggests that, similar to the smoking-associated field effect, the lung cancer-associated airway field of injury can be measured less invasively using gene-expression profiling of the nasal epithelium. Because early-stage lung cancers detected by CT screening may have distinct biologic characteristics compared to those diagnosed in patients evaluated for clinical symptoms, we propose to develop an accurate biomarker integrating expression profiling of genes and microRNA, clinical variables, and nodule characteristics in order to distinguish early lung malignancies from benign pulmonary nodules. The immediate clinical application of this test will be in the evaluation of patients with LDCT-detected lung nodules, and will allow clinicians to more accurately stratify patients to or from further diagnostic intervention. Ultimately, this test may also have utility for further stratifying lung cancer risk in patients eligible for screening prior to undergoing LDCT.

The release of small amounts of cell-free DNA into the bloodstream from dying tumor cells has been well documented in patients with various malignancies. Driven by improvements in assay technologies, such circulating tumor DNA (ctDNA) is beginning to show excellent promise as a non-invasive cancer biomarker. Because unique somatic mutations can serve as telltale marks to distinguish tumor-derived DNA in plasma, the cancer specificity of ctDNA is expected to be very high, making it an attractive candidate as a biomarker for cancer screening. Because many somatic mutations in non-small cell lung cancer (NSCLC) are found clustered with high frequency within a small set of genes, detection of ctDNA in such patients is technically more tractable. We have created a highly multiplexed, ultrasensitive assay that uses next-generation sequencing techniques to quantify low-abundance mutant DNA fragments in plasma against a large excess of background wild-type DNA. Our assay can simultaneously evaluate mutations in 40 different hotspots in up to 96 patient samples, using novel error suppression methods to optimize detection sensitivity. We have shown that the assay is capable of measuring very small amounts of mutant ctDNA in a limited set of patients with early-stage disease or with small, localized recurrences. We now aim to test the clinical performance of the assay on a larger scale. We propose to perform a case-control study comparing patients with early-stage NSCLC to individuals with a heavy smoking history or individuals having benign pulmonary nodules.

Prognosis for non-small cell lung cancer (NSCLC) is substantially better in early-stage as opposed to late-stage disease. If high-risk individuals could be effectively identified, they could significantly benefit from intensive surveillance, early detection, and prevention strategies. While smoking is a primary risk factor, only about 15% of smokers develop the disease. Consistent with a genetic predisposition, some patients have a family history of the disease and are affected at a young age, although they have never smoked. Unfortunately, NSCLC predisposing gene mutations are poorly characterized. We aim to identify novel NSCLC germline risk gene mutations. Our team members have previously identified novel risk gene mutations for several familial cancers. We have assembled a large clinically and molecularly well-characterized NSCLC case-control cohort of never-smokers, performed whole-exome sequencing (WES) on familial and early onset NSCLC probands, identified promising candidate risk genes, and externally validated selected samples. We follow a resource-conscious approach by focusing on a founder population, Ashkenazi Jews (AJ), to decrease genetic heterogeneity and to increase the allele frequencies of candidate recurrent risk mutations. We will perform WES on additional familial and early onset NSCLC probands to identify recurrently mutated NSCLC genes and cross-validate the genes using whole-genome sequencing on matching tumors. The gene candidates identified will be validated using a targeted sequencing approach in a larger non-AJ cohort. These are anticipated to provide new biological insights, serve as future chemoprevention targets, and be used in the development of a genetic diagnostic test to identify high-risk individuals.

The ALK tyrosine kinase inhibitor (TKI) crizotinib has demonstrated significant activity in patients with ALK+ lung cancer, but its efficacy is limited by the development of acquired resistance. One potential resistance mechanism involves activation of the EGFR and HER2 kinases. This activation occurs in the absence of EGFR/HER2 mutation or amplification, suggesting an alternative mechanism for up-regulation of these kinases. We discovered a novel association between ALK and the EGFR/HER2 negative regulator, MIG-6. MIG-6 is known to inhibit EGFR/HER2 kinase activity through direct binding. However, to date, there is no known link between ALK and MIG-6. In our preliminary data, we demonstrate that MIG-6 protein levels decrease after ALK TKI treatment, a process abrogated by addition of a proteasome inhibitor. Furthermore, MIG-6 transcript and total protein levels are decreased in ALK+/TKI resistant compared to isogenic ALK+/TKI sensitive cells. The decline in MIG-6 correlates with an increase in EGFR and HER2 protein levels in the resistant cells. Based upon these data, we hypothesize that ALK serves as an upstream regulator of MIG-6, and that MIG-6 suppression drives EGFR/HER2 activation in the ALK TKI resistant state. To test this hypothesis, we propose to: 1) elucidate the molecular mechanisms whereby ALK regulates MIG-6 expression, and 2) determine the mechanisms whereby MIG-6 contributes to EGFR/HER2 pathway activation in TKI-resistant ALK+ lung cancer cells. The proposed studies may lead to strategies that augment or restore expression of MIG-6 and therefore will represent a novel therapeutic approach for patients with acquired resistance to ALK inhibitor therapy.