Liquid biopsy

Metastatic non-small cell lung cancer (mNSCLC) is the leading cause of cancer-related death in the United States. Most patients with mNSCLC are treated with systemic agents, which prolong survival and improve symptoms, but are typically not curative. Although recent advancements in immune checkpoint inhibitors have revolutionized the management of mNSCLC lacking targetable driver mutations, only a fraction of mNSCLC patients benefit from immunotherapy. Emerging evidence suggests that ablative radiotherapy can augment immunotherapy responses in metastatic disease; however, strategies to optimize the synergy between local radiation and the immune system are not well defined. We are conducting a randomized phase I/II clinical trial designed to evaluate the safety and efficacy of combination immune checkpoint blockade (ipilimumab + nivolumab) plus multisite ablative radiotherapy as a first-line treatment for patients with mNSCLC. As a secondary analysis of the phase I trial, we propose to investigate biomarkers associated with treatment response. In Aim 1, we propose to compare the diagnostic accuracy of established clinical biomarkers, including PD-L1 immunohistochemistry (IHC), tumor mutational burden, gene expression profiles, and multiplex IHC in predicting response to combination ablative radiotherapy and immunotherapy. In Aim 2, we propose to characterize global transcriptional and genomic changes induced by radiation when given prior to or concurrently with immunotherapy and evaluate whether these changes are associated with treatment response. In addition, in Aim 3, we propose to validate a novel biomarker derived from alternative splicing as a predictor of radio-immunotherapy response. By elucidating the immunomodulatory properties of radiotherapy, we may be able to improve treatment responses and long-term survival in patients with mNSCLC.

KRAS mutations are the most common activating mutation in lung cancer and are identified in 25-30% of cases of lung adenocarcinoma. The clinical activity of molecularly targeted therapy in KRAS mutant lung cancer has been limited, as most prior approaches focused on downstream targets of the MAPK pathway, an approach which was largely ineffective. KRAS G12C is the most common subtype of activating KRAS mutation in NSCLC. Identification of a binding pocket of KRAS G12C enabled the development of covalent inhibitors (Ostrem et al. Nature, 2013), which selectively bind to the mutant cysteine and spare wild type or other mutant KRAS proteins (hereafter referred to as G12Ci). These drugs bind only to GDP-bound (i.e. inactive) KRAS G12C and trap the oncoprotein in its inactive state by preventing nucleotide exchange (Lito et al. Science, 2016). Of this new class of agents, two drugs (MRTX849 and AMG510) have demonstrated efficacy in early phase clinical trials, representing a remarkable breakthrough in the management of KRAS G12C mutant lung cancer. Acknowledging the limited number of patients treated thus far, response rates of 40-50% have been reported, demonstrating clinical activity far superior to prior molecularly targeted approaches for the management of KRAS mutant lung cancer. Despite this major advance it is clear that resistance to therapy, both primary and acquired, will remain a challenge and may in part be due to upstream receptor tyrosine kinase (RTK) signaling. Understanding the mechanisms of resistance to this new class of therapy is of paramount importance to improve the efficacy and durability of response, and for identifying rational combinatorial therapies for future clinical trials.

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. 

This project describes a 3-year training program for a career as a physician-scientist with the long term goal of establishing a research program within the field of translational thoracic oncology. The applicant is currently an Instructor of Medicine in the Section of Interventional Pulmonology and Thoracic oncology conducting research in developing biomarkers for the treatment selection and monitoring of patients with lung cancer. The research focus of this proposal is to develop molecular markers to better identify the subgroup of patients that will respond to PD1/PDL1 therapies in order to avoid unnecessary toxicities, reduce costs, and enable more appropriate therapies to be delivered. This goal will be accomplished in two complementary specific aims. In Specific Aim 1, a novel approach to acquire fresh tissue samples for immunophenotyping will be utilized to prospectively determine the ability of deep T cell phenotyping and functional assays to predict response to anti-PD1/PDL1 therapy using multi-parametric flow cytometry. In Specific Aim 2, a newly developed technique that enables the quantitative measurement of circulating PDL1 exosomes will be utilized to determine the association of pretreatment and on-treatment exosomal PDL1 expression levels to predict clinical outcomes in non-small cell lung cancer patients receiving checkpoint blockade. The training component of this proposal includes formal coursework, participation in a rich environment of post-doctoral lectures in thoracic oncology/tumor immunology, acquisition of advanced laboratory techniques, and individual mentoring. This project will take place under the supervision of Dr. Steven Albelda who is the Director of the Thoracic Oncology Laboratory at the University of Pennsylvania and Co-Director of the Abramson Cancer Center’s Translational Center of Excellence for Lung Cancer Immunobiology, and Dr. Anil Vachani who is the Director of Clinical Research for the Section of Interventional Pulmonology and Thoracic Oncology at Penn. Dr. Albelda and Dr. Vachani have mentored over 100 trainees. In addition, an advisory committee of distinguished scientists will provide experimental assistance, intellectual guidance, and career advice throughout the duration of this award.

Immune checkpoint blockade (ICB) has demonstrated profound effects on the anti-tumor immune response and has revolutionized the treatment landscape of non-small cell lung cancer (NSCLC). While chemotherapy has traditionally been viewed as detrimental to the immune system, recent clinical trials have demonstrated an impressive survival benefit when combining checkpoint blockade with chemotherapy (ICB+chemo) relative to chemotherapy alone. Despite this, many NSCLC patients do not achieve clinical benefit from ICB, even when combined with chemotherapy. The role of tumor mutational burden, and specifically T cells targeting neoantigens derived from these mutations, has been demonstrated in several ICB studies, however these factors have yet to be fully explored in chemotherapy-containing ICB treatment regimens. There is thus an urgent need to understand the effects of ICB+chemo on the anti-tumor immune response and to specifically evaluate how chemotherapy perturbs the phenotype and function of anti-tumor T cells. It is of paramount importance that we understand how PD-1 blockade and chemotherapy synergize to promote anti-tumor immunity in order to better stratify patients most likely to benefit from these treatments. We propose herein to perform the first comprehensive evaluation of the phenotype and function of neoantigen-specific T cells and their dynamics before and after treatment with ICB+chemo relative to ICB alone. The findings from this study will provide a detailed atlas of the immune system dynamics following treatment and will result in the development of an immunometabolic signature that will be correlated with clinical outcome. Importantly, the results of the proposed work will deepen our understanding of the immune response to ICB+chemo and will unravel the mechanisms underlying clinical response in NSCLC patients.

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.

Background: Osimertinib and other “third-generation” EGFR TKIs effectively overcome T790M-mediated resistance in EGFR-mutant lung cancer, but our understanding of acquired resistance in this setting remains limited. Importantly, the intrinsic heterogeneity of resistant cancers leads to clinical challenges. We hypothesize that in-depth study of 3rd-generation TKI resistance and the impact of genetic heterogeneity on treatment response will lead to improved therapeutic options and clinical outcomes. Study Design: We will analyze tumor biopsies, ctDNA and autopsy specimens using a variety of techniques such as next-generation sequencing, digital droplet PCR and whole exome sequencing to characterize heterogeneity in resistant cancers. In addition, we will run a prospective clinical trial to test a novel therapeutic combination designed to minimize heterogeneity and delay progression. On-treatment biopsies and ctDNA collection will be used to study how resistance develops. Impact: This project will leverage our broad array of clinical specimens, well-established translational research program and preexisting industry and academic collaborations to characterize the heterogeneity of resistance in EGFR-mutant lung cancer. We hope to illuminate clinical and biologic implications of heterogeneity, with the ultimate goal of identifying more effective therapies for patients.