Early detection

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.

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.

Better methods are required to accurately risk stratify patients with a solitary pulmonary nodule (SPN) who may undergo unnecessary procedures to exclude a cancer diagnosis. 18F-FDG Positron Emission Tomography (PET) is a widely available clinical test used to evaluate concerning SPNs and stage malignant lung nodules. It therefore facilitates clinical assessment but remains an imperfect tool for the diagnosis, localization, and prognostication of SPNs, be they infectious or malignant in nature. Furthermore, it performs well for staging lung cancer, but inaccurately categorizes patients in some cases who receive futile thoracotomies for disseminated disease, or conversely, are undertreated for localized disease. The applicant therefore plans on investigating whether circulating molecular biomarkers can increase the utility of 18F-FDG-PET imaging in clinical practice using statistical models that incorporate in vivo and in vitro diagnostics The concept here is “one-stop shopping,” where a patient can obtain routine molecular imaging and molecular diagnostics from routine blood work during an appointment that in combination increase the accuracy of a clinician’s diagnosis and facilitate the appropriate decision-making process. For this proposal we will develop a model that incorporates a circulating microRNA profile to discriminate infectious from malignant SPNs for patients along with clinical and FDG-PET data and, secondly, identify patients with malignant nodules and detectable circulating tumor cells to determine whether these cells upstage or augment clinical staging or prognosis post-operatively when compared to FDG-PET alone.

The development of non-invasive diagnostic tools to detect lung cancer at an early stage is the subject of active investigations because of its enormous potential impact on the number-one cause of cancer-related deaths. We have recently discovered by shotgun proteomics profiling 164 candidate biomarkers that are differentially expressed in stage 1 lung cancer tissue compared to non-malignant matched controls tissues and found in the circulation. Validation of these candidates by traditional methods using western blot or ELISA is a slow, low throughput, and expensive process. In this application, we propose to take advantage of the individual’s immune response to capture and detect autoantibodies directed against these 164 candidate protein targets identified by shotgun proteomics. To achieve this goal, we intend (1) to establish immune-based assays for the detection and quantitation  of lung cancer autoantibodies using an indirect ELISA; (2) to validate the diagnostic performance of our autoantibody signature in blood samples from individuals with early-stage lung cancer or matched nodule controls; and (3) to determine whether the diagnostic performance of our panel of autoantibody signatures augments the diagnostic accuracy of known predictive models for the evaluation of lung nodules. These studies may lead to the development and early validation of a new molecular diagnostic tool for lung cancer.

Recently, the National Lung Screening Trial reported a 20% reduction in lung cancer mortality and lung cancer chemoprevention trials targeting the arachidonic acid pathway have demonstrated decreases in lung cancer associate markers .These studies highlight possibilities for future reductions in lung cancer mortality through early detection and chemoprevention. Our group has previously identified smoking- and lung cancer-specific gene expression alterations in cytologically normal airway epithelial cells that can serve s a clinically relevant biomarker for the early detection of lung cancer. We propose to extend our studies of the field of injury by profiling  these cells from high-risk smokers with preinvasive lesions (dysplasia) and matched controls without lesions by RNA-Seq. We will characterize the molecular alterations (coding, noncoding, and novel RNA expression and alterative splicing) associated with the field of injury in premalignancy and identify whether these differences are similar in both current and former smokers. Next, we will develop molecular signatures that predict subsequent progression or regression of premalignant lesions as well as changes that occur as a result of progression or regression. Collectively, these signatures will aid in the stratification of high-risk smokers for chemoprevention trials, serve as intermediate markers of efficacy in these trials, and identify new targets for chemoprevention. Finally, we will explore whether these molecular signatures re associated with occult lung cancer or future lung cancer development in independent cohorts as common alterations may indicate early events in lung carcinogenesis and have additional utility as biomarkers in the early detection of lung cancer.

Small cell lung cancer (SCLC) exhibits aggressive clinical behavior and poor outcome and response to therapy. Moreover, there are no effective strategies for early detection of SCLC. Therefore, there is an urgent need to identify new strategies and markers for SCLC detection. We recently characterized the molecular airway field cancerization (FC) in non-small cell lung carcinoma (NSCLC) patients and identified airway profiles that varied spatially (n=1165) and with time (n=1395) following surgery. Moreover, we characterized the adjacent airway FC in NSCLC (n=1606 genes), a subset of which (n=299) significantly distinguished airways of smokers with lung cancer from those of healthy smokers and another (n=208) separated airways of different NSCLC subtypes. Although we previously showed that SCLCs and adjacent airways are characterized by higher level of allelic losses compared to those of NSCLC cases, the global airway FC in SCLC and its relevance to detection has not been explored. We hypothesize that the molecular airway FC in SCLC is different compared to that in NSCLC or benign lung disease and comprises biomarkers specific for early detection of SCLC. We will 1) perform global expression profiling of the airway FC and tumors or nodules in patients with SCLC, NSCLC, and benign disease for identification of airway markers specific to SCLC and 2) test identified airway mRNA markers for SCLC detection in bronchial and nasal compartments in patients with suspect lung cancer. We anticipate that our proposal, due to its unique resources and approaches, will identify novel markers for SCLC detection.