Screening

The majority of lung cancer patients present with advanced disease that is largely incurable. Efforts to improve the early detection of this disease are critical to decrease lung cancer-related mortality. Low-dose computed tomography (CT) screening in high risk patients has demonstrated reduction in lung cancer related mortality through diagnosis of earlier stage disease at the cost of high false-positive rates and the frequent need for invasive diagnostic testing when indeterminate lung nodules are identified. Detecting tumor specific somatic mutations and/or epigenetic signatures in circulating tumor DNA (ctDNA) represents an attractive non-invasive method for identifying early stage lung cancer. However, to realize this promise for lung cancer screening, where ctDNA from small tumors is miniscule, the development of sensitive, specific, and economical ctDNA based assays is critical. 
The bacterial innate immune system consisting of clustered regularly interspaced short palindromic repeats (CRISPR) and their associated endonucleases (CRISPR-Cas) are specifically adapted to recognize foreign nucleic acids. The recent identification and diagnostic adaptation of certain classes of CRISPR-Cas systems, such as CRISPR-Cas12a and -Cas13a, offer unique platforms on which to develop sensitive, specific, and economical assays for the detection of ctDNA. The goal of the current project is to develop CRISPR-Cas12a and -Cas13a based ctDNA assays that can sensitively detect somatic cancer mutations and aberrant promoter hypermethylation often found in lung cancer patients and to validate these assays in plasma samples of patients with early stage NSCLC and in discriminating between benign and malignant nodules identified by low-dose CT screening studies. 

Statement of the Problem: Cancer develops in a sequenced manner. Patches of lung cells gain the ability to multiply faster than their neighboring normal cells by acquiring mutations. These patches of cells are called “premalignant lesions" (PMLs). Some of these lesions may eventually become cancer. Knowledge about how some PMLs may develop into lung cancer is central to developing tools for Cancer Interception—interrupting the cancer development process and blocking progression to advanced metastatic disease. However, we lack effective lung cancer interception approaches due to our incomplete understanding of the earliest molecular events associated with the development of lung cancer, as well as the challenge in developing personalized tools for early detection and prevention.

Proposed Solution: The Dream Team will use a holistic approach to understand the genetics, immunology, radiological (imaging) profile, and treatment response of patients who show evidence of abnormal lung tissue that puts them at high risk to develop lung cancer. The Team has access to unique groups of patient populations (adenocarcinoma—ADC and squamous cell carcinoma—SCC) with signs of lung cancer risk, and has assembled a collection of blood and tissue specimens from these individuals. The main objective of the Team is to understand how lung cancer develops and test methods that will block the development of cancer. Their three-pronged approach to solve this problem will include:

• Creation of a molecular pre-cancer genome atlas (PCGA) of the lung with the goal of identifying which types of precancerous lung tissue require aggressive treatment and which treatments will block the development of these abnormal lesions to invasive lung cancer.
• Development of two diagnostic tools that can be directly applied in the clinic for simple, yet accurate, detection of early lung cancer: 1) the use of nasal swabs, blood tests, and imaging to confirm whether lung abnormalities found on chest imaging are lung cancer or benign lung disease. 2) blood tests that can identify patients at the earliest stages of lung cancer recurrence, thus enabling their timely and effective intervention with therapies such as those that boost the immune system.
• Development of tests to identify which individuals are most likely to benefit from a number of treatment strategies to intercept lung cancer, including emerging immunotherapies.

Statement of the Problem: Currently, low dose CT (LDCT) screening for lung cancer is recommended for high-risk individuals, defined as current or former smokers (quit within the past 15 years) with a 30-or-more pack-year smoking history, 55 to 74 years of age, and with no evidence of lung cancer. However, uptake of LDCT screening has been impeded by high rates of false-positive results, which in turn add significant physical and emotional stress to patients as well as additional healthcare costs. Since LDCT is now commonly available, lung nodules detected in LDCT scans are plentiful, but imprecise guidelines for care make it a challenge to ensure quality follow-up and expedited diagnoses for those with lung cancer. Furthermore, screening is not available for those who don’t meet the screening criteria (younger age or low smoking history) when they are the exact population among whom lung cancer rates are rising.

Proposed Solution: Leveraging expertise of academic research centers (Massachusetts General Hospital, Broad Institute, and Stanford University) and a non-profit managed-care consortium (Kaiser Permanente), the Dream Team will develop a test called the Lung Cancer Interception Assay (LCIA) to complement LDCT screening.

The proposal is novel in that it will develop complex computational algorithms to combine high-sensitivity/high-accuracy blood-based molecular biomarkers with image-based biomarkers to create the  LCIA assay. At the end of the award period, a streamlined product, the LCIA, will be ready to use in population-level definitive trials for the early detection of lung cancer.

One of the challenges for early detection and prevention of lung squamous cell carcinoma is the lack of understanding of the molecular and cellular changes that cause initiation and progression of this disease. Airway premalignant lesions are the precursors of lung squamous cell carcinoma and can be detected and monitored by autofluorescence bronchoscopy. However, many of these lesions will regress without clinical intervention, and only a subset will progress to invasive carcinoma. The goal of this study is to characterize the landscape of somatic mutations in airway premalignant lesions and to develop an early detection biomarker using targeted DNA sequencing that can predict risk of lesion progression. Ultra-deep whole-exome sequencing will be performed on a previously collected cohort of airway premalignant lesions from high-risk smokers that have been sampled over time, characterized histologically, and classified into those that are stable, regress, or progress. Significantly mutated genes, recurrent copy number changes, and mutational signatures will be identified, and a determination will be made whether they are associated with lesion histology or progression or regression status. Finally, the findings from this study will be combined with the driver genes from The Cancer Genome Atlas (TCGA) to build a targeted DNA sequencing panel. This panel will serve as a rapid and relatively inexpensive method for monitoring premalignant lesions from patients over time and can be used in a clinical trial to assess the ability of autofluorescence bronchoscopy in combination with molecular profiling to stratify patients and serve as a companion diagnostic in chemoprevention trials.

The overarching goal of the project is to develop a new technology for accurate and cost-effective lung cancer screening in current/former smokers. Lung cancer is curable if detected early. However, there are no robust screening techniques with options such as CT scans fraught with cost, harms, and a high false positive rate. Dr. Backman and his group hope to transform the clinical practice of lung cancer screening by exploiting field carcinogenesis, the concept that the genetic/environmental milieu that results in lung tumor impacts upon the entire aerodigestive mucosa including the buccal mucosa, which was referred to as a “molecular mirror” of lung carcinogenesis.

Their data show that the alteration of nanoscale architecture of buccal cells is exquisitely sensitive to lung field carcinogenesis and may serve as a robust biomarker for lung cancer. These nanoarchitectural changes can be detected in a practical and highly accurate fashion via a new technology, partial wave spectroscopic (PWS) microscopy (‘nanocytology’).

In the proposed study, they will conduct a blinded validation case-control trial to determine the sensitivity and specificity of buccal nanocytology for lung cancer. They will evaluate the ability of the test to identify patients who have lung cancer and how many patients with lung cancer would be missed using this technique. They will perform a subgroup analysis for Stage I lung cancers. They will assess the potential confounding effects of smoking frequency and time of cessation and other aerodigestive malignancies.

This project will provide the requisite data for the future definitive multicenter validation trial followed by FDA submission.

Circulating tumor cells (CTCs) are cancer cells that break away from either the primary tumor or metastatic sites. Increasingly, CTCs have been used as a readily accessible source of tumor cells (“liquid biopsy”). However, shortcomings in current CTC technologies limit downstream analyses to assessment of a selected set of nucleic acid markers. Genome and transcriptome-wide analyses of CTCs have been hampered by the low purity of CTC isolates. The complex and protracted nature of CTC capture protocols further impedes analysis.

Dr. Kulkarni and his group have developed a CTC capture technology known as Vortex Chip, which allows for rapid isolation of highly purified CTCs. In addition to standard immunofluorescence, they have generated proof-of-principle evidence that their capture technology allows for analysis of genetic material. Moreover, because the Vortex Chip isolates CTCs based on their size, their chip does not require antibody-based capture that can potentially bias isolation of CTC subpopulations. Serial CTC analyses performed in the context of treatment changes can characterize the mutational landscape of tumors as they evolve in response to therapy and thereby identify molecular predictors and drivers of treatment resistance and sensitivity to novel anti-tumor therapies.

Their project has two goals: 1) validate use of CTCs as biomarker in lung cancer and 2) explore use of CTC analysis to obtain genetic information about the tumor. They hypothesize that the unique microfluidic characteristics of lung CTCs will drive rapid isolation of pure CTC populations for analysis to characterize heterogeneity and profile therapeutic responsiveness.

Three independent principal investigators at the Fred Hutchinson Cancer Research Center with combined expertise in thoracic oncology, cancer epidemiology and prevention, and molecular diagnostics are joining efforts to respond to the Protect Your Lungs RFA. The proposed research program is focused on developing novel blood-based tests to 1) identify never-smoker subjects at increased risk of developing lung cancer (C. Li, PI), 2) detect lung cancer before onset of symptoms (G. Goodman, PI),  and 3) distinguish between benign and malignant lesions identified by CT scans (S. Hanash, PI). The research questions address important public health issues in lung cancer and build on discoveries of candidate markers by the applicant group for which we propose validation studies in this proposal. Validation studies to be conducted will take advantage of the availability to the investigators of pertinent bio-specimens consisting of samples derived from two large cohort studies (The Women’s Health Initiative and the Beta Carotene and Retinol Efficacy Trial (CARET)) and samples derived from CT screening studies. The samples available are sufficient to meet the needs of the proposed studies. The collaborative nature of the proposed research will ensure synergism and efficiency of scale to meet study objectives and fast-track the development of clinically relevant applications.

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.