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However, CT-based screening programs have been practically challenging to implement, and uptake has been slow. An alternative screening approach that has been garnering much enthusiasm is based on the development of a simple blood test that detects DNA fragments shed from tumor cells into the bloodstream. Several commercial and academic groups have been racing to develop blood tests for cancer screening based on this concept, and the field has made impressive progress. However, detection of early-stage lung cancers has remained particularly challenging, with sensitivities reaching only ~20-40% for Stage I disease. A key limitation for detection of small, early-stage tumors has been the extremely low abundance of DNA fragments bearing cancer-specific features (such as mutations) in the circulation. To overcome this limitation, Patel’s group has developed a technology that can accurately measure cancer-specific alterations in DNA which are more highly abundant (known as “hypermethylation”). In the current project, they propose to develop a predictive model to identify patients with lung cancer based on probabilities inferred from measurement of these DNA alterations. They will then further improve the sensitivity for detecting the earliest stages of lung cancer by developing an algorithm that tracks longitudinal changes in a patient’s DNA signal over time rather than relying on just a single time-point.

While it is too early in the course of this project to make any inferences on these preliminary data, there is confidence that our design was an optimal approach to answer questions about the origins of lung cancer in young people. The ALCMI trial design investigates risk factors for the development of cancer in the young population. It is also able to separate out specific NSCLC driver mutation groups, a unique feature of our epidemiological survey design, concludes that these early results suggesting that EGFR and ALK driven lung cancers may arise because of unique biological and environmental exposure risk factors. Understanding these differences may help lead to targeted treatments for these different lung cancers. Further enrollment and research will continue, and these analyses will be updated”.

To complement the results emerging from Taiwan, during the second year of the study, Dr. Sequist will collaborate with investigators from New York University to open a study of LDCT lung cancer screening for never-smoking Asian women living in the Boston area. The study involves three annual LDCTs, with clinical follow-up imaging, biopsies, and other indicated medical treatment for any suspicious lesions or lung cancers identified. Preliminary data was presented at the 2023 Annual Meeting of the American Society of Clinical Oncology.

Previously, with the generous support from Upstage Lung Cancer, the Jacks lab has developed a method to grow normal mouse lung cells in 3D culture as lung organoids. These lung organoids can be transformed in culture using a variety of techniques to induce the expression of oncogenes and loss of tumor suppressor genes. In culture, these cells display the canonical behaviors of cancer cells. For example, transformed organoids can grow in media without any growth factors. Transformed organoids can also be transferred into the lungs of healthy animals to robustly form tumors. Because transformation from normal to cancerous happens in a dish, and can therefore be acutely controlled, this system enables studies of the earliest stages of tumorigenesis. In the mouse setting, we took advantage of this attribute to look at protein changes that could be monitored in a non-invasive way early on in tumor development. Now, we plan to apply our organoid culture technique to healthy human lung cells.

Preliminary data from a small pool of human samples have shown that it is possible to grow human lung cells using our organoid system, but the propagation of these cells is limited. We plan to optimize our protocol for continuous growth and expansion of normal human lung cells. To date, no one has attempted to transform human lung organoids in a dish. Using gene-editing technology, we plan to induce mutations in the normal organoids which are known to cause cancer in human patients. We will then profile changes between the normal and transformed organoids at very early stages. Specifically, we will compare transformed and normal human lung organoids to look for factors that are differentially secreted. These comparisons would identify potential biomarkers that could be used in diagnostic tests for early-stage lung cancer. Whereas studies of primary cells in this manner are limited by material, in an organoid setting we can collect near infinite sample mass for analyses. Notably, these types of studies would be impossible with primary human cancer cells, as early on in tumorigenesis the amount of biopsy material is limited. With organoids, continuous expansion in culture allows for near infinite collection of cellular material for downstream analysis. We will then transplant the transformed human organoids into mice, extract blood samples, and see if any of these factors can be detected in the blood of tumor-bearing animals at appreciable levels early after transplantation. The Jacks lab hopes that these studies will shed light on potential biomarkers for the early detection of lung cancer.

However, up until now, liquid biopsies have proven unsuccessful in detecting various cancers. One of the challenges in bringing this diagnostic technique to fruition is the rarity of circulating tumor cells (CTCs), which are cells that break off from tumors, circulate in the blood stream and which have been demonstrated to provide a real-time genetic window into the cancer.

Researchers at the Mass General Cancer Center recently developed a new, ultrahigh-throughput microfluidic chip (the LPCTC-iCHIP) that significantly improves the sensitivity of CTC-based assays. CTCs are extremely rare, and it is estimated that there is only one CTC per billion in the bloodstream. As a result, there may be zero to about a dozen CTCs in a typical 10mL clinical blood sample. The very low number of CTCs severely limits the analytic reliability of liquid biopsy.

To address this challenge of low recovery of CTCs, our team developed a two-step leukapheresis process. Apheresis is the process of removing a specific portion of the blood while returning the remainder of the blood to the patient and is routinely used when treating a number of health conditions. This process produces an enriched “leukopak” or blood product comprised of six billion white blood cells and CTCs. Six billion cells are still too many to sort through efficiently when searching for CTCs.

This is where the LPCTC-iCHIP developed by our team comes in. First, the white blood cells (WBCs) from the leukopak are labeled with a magnetic tag, and then the entire sample is processed on the chip, which uses on-chip magnetic microlenses to remove most of the WBCs. The LPCTC-iCHIP works by removing blood cells so it is agnostic to cancer type and the team has found that it is effectively identifies CTCs with samples from any solid tumors. Our hypothesis is that the larger sample of white blood cells collected through apheresis will allow for better detection of circulating cancer cells in several types of cancer.

Advancing liquid biopsy practices with the CTC-iCHIP will increase the likelihood of detecting circulating cancer cells of several types of cancer, including breast, pancreatic and lung cancers.

The Boston Fire Department has been increasingly concerned about lung cancer risk among its members and in recent years has been recognized for its widespread efforts to increase knowledge about this problem among both scientists and firefighters. While annual low-dose computed tomography (LDCT) screening among high-risk smokers is widely recommended, insurance coverage is available for this test only in adults aged 55-77 who are current or former heavy smokers who have quit within last 15 years. These restrictions exclude an estimated 70% of the Boston Fire Department firefighters.

While it is widely known that firefighters are at increased risk of lung cancer, little is known about how their lungs appear on LDCT scan compared to similar non-firefighters. Outside of the World Trade Center First Responders, there is no published information in the medical literature about the CT radiographic features of firefighters’ lungs, or how variations in exposures affect CT scan findings. Furthermore, the risks of lung cancer among firefighters have not been specifically examined within the context of lung cancer screening trials. In a context where firefighters are increasingly concerned about their risk of lung cancer, and in which many members of the department do not meet lung cancer screening eligibility criteria, our group aims to gather more information about this central group of public servants.

Partnering with the Boston Fire Department, our research team includes investigators from the Mass General Division of Thoracic Imaging, Cancer Center, Clinical and Translational Epidemiology Unit, Center for Innovation in Early Cancer Detection and Division of Pulmonary Medicine. We are investigating the LDCT appearance of Boston firefighters’ lungs and exploring the extent to which other findings, including lung nodules, interstitial lung disease and coronary artery calcifications, are associated with occupational exposures.

Our goal is to generate critical preliminary data about lung health in firefighters by identifying a high-risk subset that may benefit from LDCT to ultimately increase access to lung cancer early detection among firefighters.

To achieve this goal, the Bhatia lab will reformulate urinary activity based nanosensors that were found to detect early-stage lung cancer (Kirkpatrick et al., Science Translational Medicine, 2020) into volatile versions that produce a breath-based readout. These newly synthesized nanoparticles will then be characterized to establish synthetic breath signatures for lung cancer versus healthy controls. Finally, to push this technology towards clinical feasibility, they will create a simple, point-of-care detection system to detect these breath signatures and classify disease.

We will conduct a case-control study to compare behaviors and exposures of interest in adolescents and young adult (AYA; defined as under 40 years old) patients with lung cancer (cases) to a gender-, age-, and race-matched group without lung cancer (controls). Through this longitudinal study (investigation done over time) we should begin to be able to answer the question: Why should a 32-year- old never smoker with no family history of cancer have lung cancer?

Innovation:  This population has not been previously and systematically studied prior to our ALCMI-003 Genomics of Young Lung Cancer project. (EoYLC) is an extension of that project.

This international study will be the first to:

• examine environmental, lifestyle, reproductive and genetic risk factors in relation to mutation specific risk (e.g. EGFR, ALK, ROS1).
• evaluate early life exposures in relation to risk of YLC, such as maternal and paternal exposures, early life diet and reproductive factors, and cumulative exposure to environmental factors, which may be associated with cancers found in the young.
• examine lifestyle or environmental factors that can modify genetic susceptibility. (Genomic Wide Associations, Genomics, Germline)

In order to discover new methods for the early detection of lung cancer, we first need to study lung cancer in its earliest stages.  Unfortunately, we currently lack the materials to do so—live patient tissue from early stage disease is not readily available for analysis, whereas most biobanked tissue is fixed, which limits the types of analyses we can perform on them.  Mouse models provide us with a rich source of tumor tissue from different stages of the disease; however, it is technically challenging to isolate cells from nascent lesions due to their small size.

In order to circumvent these issues, the Jacks lab has begun using organoids, which are miniaturized organs (in this case, the lungs) that are grown in a dish from healthy cells. The benefits of using organoids include:

• The ability to study the cancerous cells immediately after transformation, within a week or even a few days;
• Having a large sample size—the organoid can be grown to obtain sufficient material for detailed analyses;
• Having a perfect point of comparison—the ability to compare cancer cells to their healthy counterparts enables us to make confident statements about what makes cancer cells unique.

Using organoids, the Jacks lab will transform the healthy cells through gene modification to become cancerous cells. Once this occurs, they have a sample of early stage cancerous cells that can be compared with healthy cells. Their goal is to identify proteins that are expressed and secreted only in the cancerous cell group and not the healthy cell group. The expression of these proteins could then serve as a biomarker which could be tested for in blood or lung fluids to provide early, non-invasive detection of lung cancer.

Sangeeta Bhatia’s lab has sought to overcome the limitations of existing lung cancer screening tools like low-dose computed tomography (low specificity) and blood biomarkers (low sensitivity) by developing a new class of biomarkers, termed “activity-based nanosensors” (ABNs). ABNs are nanoparticles that can detect a class of enzymes called proteases, which are often overexpressed in cancer and enable tumors to grow, invade, and metastasize. ABNs are designed to travel to the site of disease and undergo cutting by a target protease to release a tiny “reporter” peptide that is small enough to travel through the bloodstream and be cleared into the urine. The lab can then measure these reporters in the urine to determine whether or not cancer is present. The goal is to develop ABNs that can detect lung cancer even earlier and can be generalized to humans—whose tumors can be extremely heterogeneous and may have mutations in many different genes.

To this end, the Bhatia Lab is working to leverage a novel organoid culture system developed in the Jacks lab to study the proteases that turn on at the earliest stages of lung tumorigenesis. Previous work performed in the Jacks Lab with generous support from Upstage Lung Cancer established that they can grow normal lung cells as organoids, and upon introduction of cancer-causing mutations, these cells can give rise to lung tumors in mice. The organoid culture system allows us to get an in-depth look at the changes that occur in normal lung cells immediately after they have been transformed into cancer cells. The Bhatia lab will use the organoid platform to determine which proteases are expressed as a result of mutations in different cancer-causing genes. With support from Upstage Lung Cancer, the teams hope to then use this information to develop ABNs that can sensitively and specifically detect these cancer-associated proteases and, ultimately, enable early detection of lung cancer in humans.

Since the beginning of the grant period, the Bhatia Lab team has tested 14 protease-detecting nanoparticles in mice bearing lung tumors with mutations in the Alk gene, which is a driver of lung cancer in humans. One nanoparticle in particular was able to detect abnormal protease activity in early-stage lung tumors, and the team sought to understand which proteases are increased in early-stage lung cancer, and what is driving this increase. The Jacks Lab team successfully generated lung organoids containing the same cancerous mutations in the Alk gene, and collected cells and proteins from these organoids.  The Bhatia Lab team then performed RNA sequencing on the organoids to identify proteases and other genes that are increased as a result of this mutation. The results of this analysis are pending, but are expected to shed light on the specific proteases that become hyperactivated in early-stage lung cancer, which can then be directly targeted to facilitate early detection of lung cancer in patients.

Although lung screening by low dose CT scan has demonstrated a significant survival advantage, the most significant risk is false positives that can lead to surgery despite what is ultimately discovered to be a benign nodule. This is due to nodules that are radiographically suspicious for cancer but upon close pathologic evaluation are found to be a benign etiology. Our proposed gas sensors are both portable and affordable for all clinics with the capacity to detect lung cancer by sensing the biomarker volatile organic compounds (VOCs) from exhaled breath. This will allow non-invasive testing of indeterminate nodules to help stratify likelihood of malignancy to reduce the number of procedures for false positives, and thereby decrease the number of complications and costs associated with procedures that carry no benefit.

Nanomaterials based sensors and trained sniffer dogs have been used for lung cancer screening by sensing biomarker VOCs from exhaled breath. However, these reported sensors are not selective, nor sensitive enough, expensive and bulky and require a meticulous breath collection process through an insulated bag, which introduces extra sources of error in the subsequent diagnosis. Our proposed gas sensors are highly sensitive, ultra-selective, hand-held with much smaller size, weight, power consumption and cost, which can sense biomarker VOCs in real time with significantly reduced error of diagnosis for lung cancer screening. We expect that our proposed gas sensors will be widely used for lung cancer screening and enable non-invasive testing of indeterminate nodules which has been challenging.

• Our sensors are hand-held, ultra-sensitive and highly selective, which provide real-time early screening of lung cancer with ultra-low size, weight, power consumption and cost.
• Our sensors will reduce the false positives noted in a lung screening program, reducing the number of benign nodules resected for suspicion of lung cancer.
• Our sensors would create the potential for lung screening in remote areas that lack a local CT scanner.

Circulating tumor DNA (ctDNA) is DNA released from dying cancer cells into the bloodstream. Individuals with early-stage lung cancer may have ctDNA in their blood, even when the cancer is localized. CRISPR-Cas technology is a novel DNA modifying tool that can be used to develop sensitive, specific, and economic ctDNA assays. Dr. Edwin Yau will develop a CRISPR-Cas-based blood test to detect ctDNA in the blood of individuals suspected of having lung cancer. While the immediate goal of the project is to evaluate this blood test in individuals who have already undergone a CT scan, the ultimate goal of the project is to develop a blood test for screening all individuals.

Lung cancer remains the leading cause of death related to cancer and the majority of lung cancer patients present with advanced disease that is for the most part incurable. Efforts to improve the early detection of lung cancer are crucial to help decrease the number of people who die from lung cancer. It has long been known that genetic information specific to cancer cells can be detected in the blood of cancer patients. Current technologies are enabling us to identify this cancer-specific genetic information directly from blood samples allowing for “liquid biopsies” that offer the potential of making cancer diagnoses directly from blood tests. However, the amount of cancer specific genetic information in the blood of patients with smaller tumors in early stages is often only present in very minute amounts that are difficult to detect. The development of new liquid biopsy technologies that can identify smaller amounts of genetic information more economically could potentially lead to blood tests that are able to detect smaller, more curable tumors and could be used for lung cancer screening. The recent identification of CRISPR-Cas systems, bacterial immune systems adapted to recognize specific genetic sequences, that can accurately detect very small quantities of specific genetic sequences are an attractive platform to develop liquid biopsy tests. The goal of the proposed project is to develop a CRISPR based blood test that can accurately identify genetic sequences specific to lung cancer and can accurately identify patients with early stage lung cancer.

Circulating tumor DNA (ctDNA) is DNA released from dying cancer cells into the bloodstream. Individuals with early-stage lung cancer may have ctDNA in their blood, even when the cancer is localized. CRISPR-Cas technology is a novel DNA modifying tool that can be used to develop sensitive, specific, and economic ctDNA assays. Dr. Edwin Yau is developing a CRISPR-Cas-based blood test to detect ctDNA in the blood of individuals suspected of having lung cancer.

During the first year of his award, Dr. Yau has developed a blood test to detect four of the most common mutations found in non-small cell lung cancer: KRAS G12C, KRAS G12V, EGFR L858R, and EGFR Exon 19 deletion comprising between 40%-50% of mutations found in adenocarcinoma, a type of non-small cell lung cancer. His test was able to detect these mutations in 18 out of 20 samples, suggesting that his test is sensitive and specific. Dr. Yau’s laboratory is now proceeding to validate this test in a larger patient sample.

Genotype-directed targeted therapies are revolutionizing cancer care. As cancer genotyping grows increasingly important in the delivery of personalized patient care, a major limitation is the availability of tumor biopsy specimens for comprehensive assessment of cancer biology. Thus, noninvasive techniques for tumor genotyping will be needed if we are to realize the potential of genotype-directed cancer care. Through a collaboration between clinical-translational investigators and laboratory scientists, we have been studying two approaches for noninvasive quantitative genotyping of cell free plasma DNA (cfDNA). First, we have validated a droplet digital PCR (ddPCR) assay which emulsifies cfDNA into ~20,000 droplets and allows rapid detection and quantification of common EGFR & KRAS mutations in plasma. Second, we have recently developed a targeted next-generation sequencing (NGS) assay which uses a bias-corrected capture and analysis approach to maximize the efficiency of rapid NGS. A pilot of this assay has shown that it can, blinded to the tumor genotype, accurately detect mutations, insertions, rearrangements, and amplifications in plasma. Our ongoing research now studies these two complementary approaches to characterize the strengths and weaknesses of each as tools for personalizing the care of advanced NSCLC. In the proposed research project, we focus on early stage lung cancer – developing highly sensitive assays for detecting cancer DNA when no cancer is evident on scans. These assays differ somewhat from those for advanced cancer genotyping as they sacrifice broad coverage of targetable variants and focus on deep sequencing of genes commonly mutated in cancer. Such assays offer an opportunity to detect lung cancers early, before they spread, and increase the chance of achieving a cure.

This research was presented this year at the Annual ASCO meeting in Chicago, 2015. It will also be presented at World Lung in Denver, CO at the IASLC, World Lung Cancer Meeting.
In breast cancer and leukemia, research has demonstrated that diagnosis at a younger age is associated with a distinct biology and natural history; however, lung cancer biology in the young (under 40) has never been systematically studied. Technological advances, such as next-generation sequencing, now provide tools to study lung cancer genetics in younger patients who typically do not express common mutations. ALCMI will use this sequencing to determine whether lung cancers in young people harbor a distinctive spectrum of genetic mutations requiring individualized management. Studying this biologically unique population may also identify new genetic sub-types needing targeted treatment strategies. This exciting study leverages ALCMI’s network of cancer centers/research management systems and the patient outreach of ALCMI’s “partner” organization, the Bonnie J. Addario Lung Cancer Foundation.https://go2foundation.org/https://go2foundation.org/https://go2foundation.org/https://go2foundation.org/

Early, accurate diagnosis of lung cancer is fundamental to improving patient survival. Tissue biopsy is key to early diagnosis in order to determine if a lung nodule is malignant or benign. Unfortunately, low-risk methods of biopsy, such as bronchoscopy, are often not able to adequately sample targeted nodules when they are small and/or difficult to navigate to. If a diagnosis cannot be made, patients must undergo repeat biopsy or even surgery, which increases risk and delays diagnosis and therapy. This project aims to dramatically improve lung cancer diagnosis on low-risk biopsy using cutting-edge optical imaging tools in combination with navigation techniques to provide (1) real-time, intra-procedural assessment of biopsy site locations to ensure adequate tissue sampling and (2) large-volume “virtual optical biopsy” of nodules for diagnosis as a complement to tissue biopsy. This project will result in a powerful new bronchoscopy tool that could reduce unnecessary risky procedures, eliminate delays in diagnosis, and allow earlier therapy initiation.

TECHNICAL ABSTRACT

Inadequate biopsy yield is a significant limitation to early lung cancer diagnosis and is a two-pronged problem: (1) Early-stage lung cancers are not adequately targeted during biopsy due to their small size and (2) even if a nodule is adequately targeted, often there is insufficient tumor in the biopsy to make a definitive diagnosis and/or accomplish all diagnostic testing for patient care. Optical coherence tomography (OCT) is a high-resolution imaging modality that provides virtual visualization of tissue volumes orders of magnitude larger than biopsy without tissue removal. Dr. Hariri’s group has developed OCT catheters compatible with standard bronchoscopes and demonstrated that OCT can identify lung nodules and assess pathologic features of lung cancer with high sensitivity/specificity. This project’s aims are to dramatically improve diagnostic capability in lung cancer using large volume OCT to provide (1) virtual intra-procedural evaluation in real-time (VIPER) of biopsy site location and 2) large volume virtual optical biopsy for subsequent pathology diagnosis as a complement to tissue biopsy. In Aim 1, they will assess if in vivo OCT VIPER biopsy assessment increases tumor yield on bronchoscopic biopsy in a powered, blinded clinical study. In Aim 2, they will use the data obtained in Aim 1 to determine the diagnostic accuracy of large volume OCT optical biopsy with tissue biopsy as compared to tissue biopsy alone. This project will result in clinical translation of a powerful, robust new optical bronchoscopy tool that could reduce unnecessary diagnostic procedures, eliminate delayed diagnoses, provide earlier intervention, and improve patient morbidity/mortality.

CT scans are used to diagnose lung cancer. When lung CT scans show lesions that are difficult to access or too small to biopsy well, many doctors wait and see whether the lesion grew before doing the biopsy.  In this way, they are saving patients the expense and discomfort of repeat biopsies and unnecessary surgeries. However, if the lesion is cancerous, watching and waiting could delay treatment. Dr. Hariri has been implementing a well-known technique called Optical Coherence Tomography (OCT) to help guide biopsies. OCT measures back-scattered light from tissues to create high-resolution images. Because the light is so gentle, OCT is routinely used for light-sensitive procedures, such as conserving valuable art pieces and visualizing patients’ retinas.  In preliminary studies, Dr. Hariri and her colleagues found that they could pass a tiny OCT probe through the biopsy needle to get a quick look at the surrounding tissue and assess needle placement to ensure an optimized biopsy sample. This method could potentially allow pathologists to consistently get the tissue they need for accurate diagnoses, even if the lesions are small or difficult to access. In addition, this process could allow pathologists to be in the room during the biopsy and to study more of the patient’s tissue in real-time without having to take additional samples.

The goal of this project is to translate a state-of-the-art nanoscale imaging technology, nanocytology, into an inexpensive and accurate lung cancer screening method among current/past smokers via an automated analysis of nanoscale morphology of buccal (cheek) cells obtained by a simple check swab. Dr. Backman and his group envision buccal nanocytology becoming a prescreen for lung cancer that is simple and reliable enough to be performed by a primary care physician or dentist as part of an annual exam of current or past smokers. If the buccal PWS is positive, a patient can be offered more expensive/invasive diagnostic tests such as low dose CT.

This is analogous to the other two-tier screening approach: Pap smear –> colposcopy paradigm, where the initial low-cost, patient-compliant screening test is applied on the entire screening population and used to identify a small subset of these patients who would actually benefit from more extensive examination. This two-tier screening strategy has been highly successful in reducing cervical cancer mortality by 90%. Their goal is to develop a similar approach for lung cancer screening.

TECHNICAL ABSTRACT

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.

Dr. Backman’s and his research team have developed and clinically evaluated a low-cost, accurate and minimally invasive prescreening technique for lung cancer by nanoscale analysis of the buccal (cheek) cells. This test will be performed by a primary care physician on current/past smokers as part of their annual physical exam. The grant project involved the development of a self-contained (easy-to-use) high-throughput PWS (HT-PWS) instrument, verification of nanoscale sensitivity of the HT-PWS instrument, optimization of the sample processing parameters and finally clinical validation. This assay will now be tested in a larger group of patients to check how sensitive and specific the test is.

Over the last few decades, cancer biologists have discovered that one of the ways cancer cells endure is that they receive “survive and grow signals” from surrounding cells in what is called the “tumor microenvironment.” Understanding these signals could provide new ways to treat cancer. This team recently discovered a mechanism by which tumor cells and the surrounding microenvironment (also called the stroma) communicate to increase tumor cell growth. CLCF1, a protein produced by cells in the stroma, is received as a growth signal by tumors cells expressing a “receptor” for this protein (termed CNTFR). This research project will capitalize on this knowledge to develop a new drug (called a “receptor decoy”) that blocks the interaction between CLCF1 and CNTFR. This drug is not a chemical but rather a small protein. The team will develop a variant of this protein that is highly effective in blocking the CLCF1-CNTFR interaction, and will test the efficacy of this receptor decoy in primary human tumors that are propagated in mice. These tumor samples are obtained directly from patients and are better models for evaluating cancer therapeutics than established cell lines that have been grown in culture for many years and have thus adapted to artificial conditions not requiring a tumor microenvironment. The team will also develop ways to predict which lung cancer patients are likely to respond best to this protein-based therapy.

TECHNICAL INFORMATION

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.

The neighborhood of a cancer cell is called the “tumor microenvironment.” Apart from the cancer cell itself, this neighborhood has other cells, such as fibroblasts and immune cells, and blood vessels that feed the cancer. Dr. Sweet-Cordero’s team is especially interested in cancer-associated fibroblasts (CAFs), special types of cells in the tumor microenvironment. CAFs make high amounts of a protein called CLCF1. His laboratory discovered that some non-small cell lung cancers (NSCLCs) produce large quantities of a protein called CNTFR. This protein is found on the surface of NSCLC cells. CLCF1 made by the CAFs sticks to CNTFR on the lung cancer cells and provides “don’t stop growing” signals. Dr. Sweet-Cordero and his collaborator, Dr. Jennifer Cochran from Stanford University, has developed small peptides (small molecules that contain a few amino acids) to block the interaction between CLCF1 and CNFTR. These peptides have shown promise in blocking the growth of NSCLC cells in models. Dr. Sweet-Cordero is planning on translating these findings to the clinic.

Cigarette smoking is a major cause of lung cancer. Finding lung cancer earlier in smokers by using low-dose CT followed by appropriate treatments can save lives. However, low-dose CT always causes overdiagnosis. The overdiagnosis requires additional medical evaluation and has negative consequences such as anxiety, additional medical costs, and other harms to the individuals who don’t have cancer. Therefore, noninvasive methods that can improve CT for accurately finding early lung cancer are urgently needed. To this end, we previously developed a panel of sputum-biomarkers (NBC News and AACR Breaking News) that can improve CT’s performance for lung cancer early detection. Yet these biomarkers are still not efficient for clinical use. Herein, we will use the most advanced technique to develop new and effective biomarkers to improve CT for finding lung cancer at early stages in smokers. Future use of the biomarkers together with CT will enable effective treatments to be immediately initiated for lung cancer, and hence decrease lung cancer deaths. It will also avoid harmful treatments to individuals without cancer and cut the tremendous burden of lung cancer on patients, families, and societies. Therefore, this project will strongly support the mission of LUNGevity Foundation.

TECHNICAL INFORMATION

Screening smokers with CT can detect lung cancer at relatively early stages, and thus reduce mortality. However, CT screening for lung cancer has a poor specificity, often resulting in unnecessary treatments to smokers who have benign diseases. The objective of this proposal is to develop genomic- and non-coding RNA (ncRNA)-based biomarkers in sputum that can complement CT for lung cancer early detection in smokers.  Proposed aim 1 is to define sputum ncRNA signatures of early-stage lung cancer using next-generation sequencing (NGS). Proposed aim 2 is to optimize a panel of sputum ncRNA biomarkers with CT for the early detection of lung cancer. Proposed aim 3 is to integrate our previously identified genomic biomarkers and the newly optimized panel of ncRNA biomarkers with CT for the early detection of lung cancer in independent case-control series. Future use of the biomarkers in clinical practice for screening smokers for lung cancer will increase CT’s specificity, and hence reduce harmful and unnecessary treatments to many smokers who have benign diseases. Integrating the biomarkers with CT could assist making decisions for the management of abnormal findings in the lungs and enabling effective treatments to be immediately initiated for lung cancer. The study will translate the strengths of novel ncRNA profiling data developed from NGS into clinical settings by using a simple and cost-effective approach, and thus bridge the gap between basic research and patient care.

All cells produce molecules called RNA, which are usually “copied” to make proteins. However, not all RNA is copied into proteins. Dr. Feng Jiang is studying one such group of RNAs, called non-coding RNA (ncRNA). His team discovered that sputum of lung cancer patients contains cells that make unique ncRNAs. They are using an innovative technology called deep sequencing to study these unique ncRNAs and develop a signature for the early detection of lung cancer. This approach has the added advantage of not requiring a surgical biopsy—ncRNA extracted from cancer cells in sputum can be studied. Dr. Jiang is confirming the validity of these new biomarkers in early-stage cancer patients who have lung nodules in a CT scan. ncRNAs are a new avenue to explore for the early detection of lung cancer.

The treatment of lung cancer has been revolutionized by the discovery of specific targeted therapies such as erlotinib or gefitinib for EGFR-mutated lung cancer and crizotinib for ALK-translocated lung cancer. These successes have taught us that lung cancer is not just one disease, but a multitude of different diseases, best defined by the specific tumor genetic changes that are driving tumor growth and that can serve as targets for therapy. At Massachusetts General Hospital, we have been performing tumor genetic testing via SNaPshot, a panel of known oncogenic mutations, since 2009. We have found that approximately 40% of our patients do not have an identifiable mutation. For these patients, identifying what tumor genetic changes are driving their cancer will be critical to develop effective targeted therapy. This proposal therefore is focused on those patients who did not have an identifiable tumor mutation, with the hope that we can discover new targets for effective therapy. We plan to do this by two methods: First, we will perform whole exome sequencing, i.e. sequencing the coding regions of the tumor genome, on the tumors of patients who did not have any identifiable tumor mutations. Second, we will screen for chromosomal rearrangements involving tyrosine kinases. Tyrosine kinases are of particular interest in cancer, as they play a role in cell growth and proliferation, and more importantly are potentially “druggable.” With this two-pronged approach, we hope to identify novel tumor-related genetic changes that will lead to new targeted therapies for lung cancer.

TECHNICAL INFORMATION

Targeted therapies such as erlotinib or gefitinib for EGFR-mutated lung cancer and crizotinib for ALK-translocated lung cancer can yield remarkable responses and significant clinical benefit. Other mutations in genes such as K-RAS, BRAF, and PI3K, among others, are also being actively investigated as targets of new drugs in clinical trials. However, a substantial number of patients (~40%) have no mutations when tested with a panel of known oncogenic mutations. The aim of this research proposal is to identify novel somatic genomic alterations in lung cancer, using an existing tumor bank of lung cancer cases with known clinical and tumor genetic information. We will focus on those lung cancers that are known to be wildtype on SNaPshot and ALK FISH (fluorescence in situ hybridization) (i.e., negative for over 50 tested oncogenic mutations), and perform both deep sequencing of exomes and a comprehensive search for translocations involving kinases. We believe that this effort will identify novel tumor genomic changes that can be targets for therapy.

The goal of precision medicine is to match cancer patients with the right therapies. Targeted therapies have shown great promise in this regard. However, 40% of patients with lung adenocarcinoma do not test positive for a druggable target. Dr. Rebecca Heist is studying this group of patients. Using cutting-edge DNA sequencing technology, she has identified two new changes in the DNA of lung cancer patients: a mutation in the MERTK gene and a NRG1-CD74 fusion (NRG1 and CD74 that are normally produced as separate proteins but get fused in cancer cells to form one big protein). Both these changes are treatable with existing drugs. In addition, Dr. Heist has been involved in the development of drugs for rare mutations in lung adenocarcinoma. Her research has recently led to the approval of capmatinib, a drug targeting MET Exon 14 skipping mutations. Dr. Heist’s strategy is setting the stage for the discovery of new “druggable” targets that allow for customized treatments to adenocarcinoma patients.