Basic Research:
The following projects emerged from years of interdisciplinary discussion and collaboration among researchers at the Brigham and Harvard Medical Area interested in mesothelioma. The strategic approach has been to build an integrated research effort among multiple disciplines each contributing to a different aspect of the program. All projects share access to patients, clinical samples, and a central database of patient and research data. Only patients who consent will be studied. Each project is ultimately designed to improve patient therapy and outcome.
The Epidemiology Project will collect patient history and exposure data to assess genetic susceptibility factors for development of malignant pleural mesothelioma (MPM). The basic science and preclinical projects (Growth Regulatory Lesions in Mesothelial Oncogenesis, Kinase Targets and Genomic-based Projects) will contribute to the biology of MPM development, discover and validate molecular markers or targets, and develop targeted therapies. The Clinical Management Project will attract patients, track outcome data, supply patient blood and tissue samples to the other projects, and assess new options for diagnostic tools, chemotherapy, surgery and post-treatment care. Synergistic progress is expected from the coordinated efforts of the proposed studies.
Individual Projects:
1. IMP Researchers Develop a Methodology for Identifying Mesothelioma-Related Mutations: A Major Step toward Advancing Treatment
Scientists of the International Mesothelioma Program (IMP) have discovered the unique genetic mutations of multiple mesothelioma tumors from individual patients. They found that each tumor had its own unique mutational profile – its own genetic fingerprint – and in the not-too-distant future, the ability to obtain this unique genetic fingerprint will improve therapeutic decisions for every cancer patient.
Tumor samples from four mesothelioma patients were collected at resection, and a new technique, developed by IMP researchers and known as microaliquoting, was employed to select the optimal part of each tumor sample for testing.A cutting-edge, high-throughput DNA sequencing technology was used to search all of the expressed tumor genes (the transcriptome) for evidence of mutations likely to be involved in making mesothelioma a difficult cancer to treat.
This is the first time researchers anywhere have identified in an unbiased manner all of the mutations associated with any cancer. Currently, genetic studies of tumors focus on known mutations in genes that may or may not even be expressed (have functional significance). The IMP researchers’ approach – to sequence all expressed genes without any preconception as to which might be more or less important – facilitates the discovery of previously unknown mutations in genes not before linked to cancer that are associated with mesothelioma and perhaps other cancers, as well.
In the four tumors studied, 15 mutations of multiple types were discovered. Three to four new mutations, none of which had been implicated in cancer in previous studies, were found in every tumor on genes that could be causally related to cancer. Although every tumor had its own unique mutation profile, further studies found that some of the mutations also were observed in tumors from other malignant pleural mesothelioma patients, making it that much more likely that these novel genes/mutations are important in mesothelioma.
The findings were published in early 2008 in the prestigious Proceedings of the National Academy of Sciences. Raphael Bueno, MD, IMP research co-director, said, “There are probably several hundred unique mutations associated with mesothelioma. Our goal is to identify and validate as many of them as possible in an effort to better understand what signaling pathways are involved and develop therapies that target those pathways.”
The IMP’s genomic analysis team partnered with 454 Life Sciences to use an ultra-high throughput technology known as pyrosequencing that permits analysis of hundreds of millions of nucleotide bases (the building blocks of DNA) per machine per day, more than 100 times more bases than conventional approaches in the same time frame. They also collaborated with the National Center for Genome Research to develop software tools to manage the billions of DNA sequence data points.
IMP Program Director David J. Sugarbaker, MD, lead author of the paper, said, “One truly encouraging aspect of our findings is that after spending a year and a half to develop the methodology and software for the pipeline, new tumors can be analyzed in a relatively short time, over the course of a month. Ultimately, this technique opens the door to individual analysis of every tumor and treatment decisions based on the specific genetic characteristics of the tumor. It heralds personalized therapy for every mesothelioma patient.”
Bueno added, “Currently, it is not possible to form any conclusions about the significance of any mutation, but as more and more tumors are sequenced through our pipeline, the discovery of tumor mutations that underlie mesothelioma and other cancers will accelerate. Down the line, this may mean that we will be able to sequence a patient’s transcriptome, identify those pathways that are perturbed, and know which drugs may be most beneficial.”
IMP scientists have made this database and their analysis available online and have created software tools that allow other researchers to access and query it. They hope that sharing data will speed the pace of discovery and improve therapy for all mesothelioma patients. Bueno said, “We want to cure this cancer. If other scientists around the world can use our data to that end, we are happy to share our work with them.”
The data are available at this site, where researchers who wish to access this database can obtain a username and password.
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2. Novel Therapeutics Laboratory Identifies Innovative Approaches to Mesothelioma Treatment
The International Mesothelioma Program’s (IMP) Novel Therapeutics Laboratory seeks to elucidate the mechanisms of action both of mesothelioma and of the chemotherapeutic agents used to combat it, with the goal of identifying opportunities to improve treatment. Over the last two years, IMP Novel Therapeutics Laboratory Director David J. Sugarbaker, MD, and Co-director Mor-Li Hartman, PhD, have been investigating the effects of cisplatin and rapamycin, alone and in combination, on six mesothelioma cell lines.
Cisplatin is the drug most commonly used in clinical practice to treat mesothelioma. Cisplatin, however, has multi-organ toxicity combined with a limited therapeutic capacity, and identification of additional agents to enhance its action is desirable. Rapamycin is a well-established inhibitor of an essential protein that is part of a signaling pathway known to be involved in malignancy and has been suggested as a potentially beneficial chemotherapeutic agent.
Results in cell lines indicate that the combination of rapamycin and cisplatin significantly increased cell death compared with either drug alone. Further, in at least one cell line, pretreatment with rapamycin may enhance the effect of combined treatment. Hartman said, “These results present the basis for potential clinical evaluation of rapamycin and cisplatin combination therapy for mesothelioma patients.”
Tamara Tilleman, MD, PhD, Medical Director of Clinical Research in the Division of Thoracic Surgery at Brigham and Women’s Hospital, said, “Surgeons and researchers in the Division of Thoracic Surgery wrote a protocol for a clinical trial based on Dr. Hartman’s and Dr. Sugarbaker’s findings. This protocoldescribes an evidence-based medical treatment combining our best scientific and medical expertise and the current medical evidence regarding treatment of malignant pleural mesothelioma.”
In the coming years, Novel Therapeutics Laboratory investigators will continue the search for agents that may provide the basis for innovative, safe, and effective therapies. Three specific projects are:
· analysis of treated cells to discover what genes and proteins are responsible for the action of chemotherapeutic agents and to better understand the mechanism of each drug alone and of both drugs in combination
· characterization of biomarkers for single agent and combined treatment. If these biomarkers can be validated in clinical use, doctors can use that information to tailor therapy to each individual patient
· investigation of additional chemotherapeutic agents. Cisplatin and rapamycin act in different ways and have distinct mechanisms of action on mesothelioma cells. Researchers plan to test additional drugs that would inhibit additional signaling pathways and potentially result in increased tumor cell death. They also will analyze whether by adding other drugs, they can reduce the concentration of cisplatin and minimize its toxic systemic effects.
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3. IMP Researchers Use Novel Testing Model to Study Effects of New Mesothelioma Drugs
International Mesothelioma Program (IMP) researchers are poised to begin investigation of a promising new drug for mesothelioma, using a novel, more clinically relevant model for testing response to chemotherapeutic drugs.
Over the past three years, the IMP has provided substantial support for a project to study, in cultured mesothelioma cell lines, the drug bortezomib (Velcade®) and all cancer-related proteins within the cell that bortezomib interacts with and affects. Bortezomib is an FDA-approved agent that is being used in the treatment of (and studied in the context of), other cancers, but has not been studied extensively in mesothelioma.
Bortezomib is a promising drug for investigation because it blocks activation of certain inhibitors of programmed cell death known as IAPs, or inhibitor of apoptosis proteins. IAPs, in turn, are responsible for substantial drug resistance in mesothelioma.
Gavin J. Gordon, PhD, co-director of the Thoracic Surgery Oncology Laboratory at Brigham and Women’s Hospital, and his team of researchers successfully demonstrated that bortezomib increases the ability of the chemotherapy drugs cisplatin and pemetrexed (Alimta®) to cause cell death in mesothelioma cell lines. This suggests that bortezomib alone or in combination with standard chemotherapy may improve outcomes for patients with malignant pleural mesothelioma.
According to Gordon, “These findings justify further investigation of bortezomib in the treatment of mesothelioma.” He and his team plan to study the effect of the drug in tumor fragments called spheroids.
Compared to cultured cell lines, actual tumor cells in tumor fragments behave more naturally. In an actual tumor fragment, other cell types are present, the extracellular matrix is present, and the cells communicate in a three-dimensional manner throughout the tumor. All these factors influence patterns of gene expression and drug sensitivity of tumor cells.
In developing cancer treatments, it is not unusual to see drugs that work well in cell lines but not in humans. Testing potentially useful drugs in spheroids may provide a valuable intermediate step between testing in cell lines and clinical trials in patients.
IMP Director David J. Sugarbaker, MD; Gordon; and Courtney Broadus, MD, Professor of Medicine, University of California at San Francisco, collaborated in the development of the spheroid model for testing drugs for mesothelioma. Broadus has tested other mesothelioma drugs using this model, and according to Gordon, “Bortezomib will be the first drug in the East Coast spheroid pipeline.”
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4. Epidemiology of Malignant Mesothelioma – Karl T. Kelsey, M.D.
e goal of this project is to conduct a comprehensive epidemiologic study of MPM to investigate the underlying disease etiology using both a case control and a case-series design. The case control study includes all incident cases of MPM identified through the Brigham and Women’s Hospital and the Dana-Farber Cancer Institute. This project will recruit the base population for the entire program. We will administer a detailed risk factor, demographic and clinical questionnaire to both cases and controls that will be used to identify exposures and traints that increase the risk of MPM.
Tumor profiling, including assessment of epigenetic silencing, will be used to further our understanding of the mechanism of action of asbestos, asbestiforma materials and other exposures that may contribute to this disease.
Thus, the objectives of the proposed study are to further the understanding of the extent and etiology of malignant mesothelioma.
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5. Innovative Test Will Help Physicians Customized Mesothelioma Therapy
Researchers in the International Mesothelioma Program’s Thoracic Surgery Oncology Laboratory at Brigham and Women’s Hospital have developed and validated a gene-ratio test that uses expression levels of four genes associated with mesothelioma to predict whether an individual patient is likely to benefit from standard trimodality mesothelioma therapy.
The four genes used in this test were identified five years ago by researchers in the Thoracic Surgery Oncology Laboratory using microarray profiling analysis to identify among tens of thousands of genes expressed by individual mesothelioma tumors those which are differentially expressed in tumors from patients with widely divergent clinical outcomes. These four genes were meaningfully expressed in tumors from patients spanning a broad spectrum of ages, genders, ethnicities, and tumor subtypes.
The test was confirmed first in a large number of independent patient tumors from a retrospective database, and then validated in a prospective clinical trial (the current scientific standard). When combined with other patient-specific information, the test allows surgeons to predict with high certainty which patients will respond favorably to surgery and which should consider more aggressive experimental therapy.
In the prospective clinical trial, the test successfully predicted clinical outcome (i.e., survival) and, encouragingly, proved to be highly repeatable and reproducible, which is an important consideration since mesothelioma tumors are large and heterogeneous. Raphael Bueno, MD, co-director of the Thoracic Surgery Oncology Laboratory, said, “This information will be valuable in driving physicians’ recommendations for therapy. For patients who have a predicted poor outcome, we know that the current treatment algorithm of surgery, radiation, and chemotherapy may be inadequate, and we may choose to customize our treatment, perhaps adding neo-adjuvant chemotherapy or another therapeutic agent, to address the patient’s unique circumstances.”
Bueno intends to offer this test first in the context of a clinical trial and then to all patients. A small tissue sample will be taken either through a minimally invasive procedure called thoracoscopic biopsy, or through an even less invasive percutaneous (through the skin) procedure. This percutaneous test is the subject of a sub-investigation to evaluate the efficacy of ultrasound-guided fine-needle biopsy as compared with the thoracoscopic approach and to define by radiological criteria the subset of patients in which the percutaneous approach is feasible.
The samples, however derived, will be tested and patients can be assigned to a predicted-good-outcome or predicted-poor-outcome group. Patients and their physicians can then use this prognostic information to design the most appropriate treatment strategy.
Gavin J. Gordon, PhD, co-director of the laboratory, said, “We don’t know why one group of patients is likely to have good outcomes and another is not. We don’t know whether the genes are driving response to therapy, or are merely reflective of response; we don’t know if they play a mechanistic role. We just know that these genes are the beacons that tell us, ‘this patient is likely to have a good response, and this patient is unlikely to do well.’ The next step is clearly to determine what these genes are actually doing so that next-generation therapies can be developed.”
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Among the goals of the Thoracic Surgery Oncology Laboratory are the identification of genes that are abnormally expressed in mesothelioma and development of better treatments for patients. Researchers are increasing their portfolio of gene-based tests, and next on the horizon are a diagnostic test for mesothelioma and a prognostic test for response to chemotherapy.
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6. Kinase Targets in Mesothelioma - Jonathan A. Fletcher, M.D.
The aims of the proposed studies are to make therapeutic advances in mesothelioma by characterizing kinase activation mechanisms in mesothelioma patients and by evaluating clinical regimens designed to circumvent these mechanisms.
This project will identify and evaluate activated receptor tyrosine kinase proteins as novel therapeutic targets in mesothelioma. Oncogenic receptor tyrosine kinases play key roles in the pathogenesis of many types of cancer, and they have emerged recently as compelling therapeutic targets, particularly when activated by genomic mutations resulting in kinase sequence alterations or overexpression. These therapeutic developments have prompted the large-scale validation of small molecule, peptide, and immunological inhibitors of various receptor tyrosine kinase proteins with suspected roles in tumorigenesis. EGRF and MET have been implicated as potential receptor tyrosine kinase targets in mesothelioma, and our own preliminary proteomic and in vitro studies suggest that AXL can also be activated strongly in mesothelioma.
The overall aim of this project is to discover oncogenic kinase targets (both receptor and non-receptor tyrosine kinases), and to determine whether such kinases serve as appropriate therapeutic targets for patients with mesothelioma. This project is highly translational in that the objective is to identify drug targets that are evaluable in the near-term in clinical trials.
Genomic-based Prognosis, diagnosis, and Development of Novel Therapeutic Strategies in Mesotheleioma - Raphael Bueno, M.D.
Malignant Pleural Mesothelioma (MPM) is a highly malignant neoplasm for which therapy is inadequate. We have studied gene expression in MPM with microarrays and developed diagnostic and predictive tests for patients undergoing surgery for this disease using the gene ration methods. We also discovered that the anti apoptotic gene IAP-1 mediates chemotherapy resistance in mesothelioma cell lines. In this project we plan to test our diagnostic and prognostic tests in tissue biopsies obtained from patients enrolled in the clinical projects of this program project. We hope to identify the best predictive and diagnostic test for each of the therapies and at the same time discover new targets for research.
We will also work on elucidating the mechanism by which IAP-1 mediates cisplatin resistance in MPM cell lines. We will determine if any additional anti apoptotic genes mediate such resistance and then construct cell line models to test inhibitors of involved pathways with the hope of identifying new targets for therapy. This approach will also be used for other potential targets discovered during our microarray analysis.
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7. Innovative Technique Enhances Mesothelioma Research
Scientists in the International Mesothelioma Program’s mesothelioma tumor and tissue bank have pioneered an innovative method for characterizing frozen tissue samples with an unprecedented level of precision.
This technique, known as microaliquoting, recently was described in the journal Biotechnic and Histochemistry, and lead author William Richards, PhD, operations director of Brigham and Women’s Hospital Tissue and Blood Repository, said, “This technique represents the next generation of prospective quality analysis for frozen tissue samples and will provide valuable information for scientists studying the genetic basis of diseases, including mesothelioma.”
Most genomic research is carried out using cell lines, which may be derived either from actual tumors or from normal cells that have been manipulated to behave like malignant cells. Using cell lines in research has some advantages – researchers can make as many cells as they need, and every cell is a tumor cell.
But cell lines exist in two-dimensional space, independent of the lymphocytes, fibroblasts, and other cells contained in actual tumor tissue. Richards said, “Cell lines do not participate in the elaborate molecular dance that is characteristic of living organisms, and that is why they do not always behave like actual tumor cells.”
Tumor tissue is far more complex than cell lines. It contains mixed cell types, and different areas of the tumor contain different mixtures of cell types in different proportions. Furthermore, the mix of cells within a given sample can change dramatically over very short distances, especially in mesothelioma tumors. Some areas will contain a preponderance of normal cells and others will be dominated by malignant cells.
The conventional technique for characterizing a tissue sample for research is to describe the cell content of a slice obtained from one, or at most two, ends of the sample. However, the intervening tissue may have a very different cell make-up from the two end slices, and therefore this method may not sufficiently account for the heterogeneity of the sample.
The microaliquoting technique involves making very thin slices throughout the frozen tissue sample and examining them under a microscope to determine the percentage of tumor cells. “Microaliqoting allows us to characterize each part of the frozen sample with much more precision, and it enables us to fulfill specific requests from researchers – for example, a request for samples that are highly enriched in tumor cells or perhaps a sample with a specific range of fibroblasts,” said Richards.
It is important for researchers to know the exact cell content of a tissue sample in order to correctly interpret results. “Normal cells increase the ‘noise’ around the signal and make it more difficult to detect the signal you are looking for. Microaliquoting is a way of increasing the signal-to-noise ratio,” said Richards.
Microaliqoting also is able to provide the level of tissue characterization needed to validate gene-based diagnostic and prognostic tests. This information will allow test developers to establish a quality threshold for clinical samples, that is, a range of specimen parameters in which their test is accurate, and a range in which their test will be fooled. It is a way of “testing the test.”
According to Richards, the microaliquoting technique, although very labor-intensive, uses technology readily available in most laboratories. “Because it offers a more precise degree of tissue characterization, it can play a valuable role in improving treatment for mesothelioma and other diseases.”
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The International Mesothelioma Program’s tumor and tissue bank – part of the Tissue and Blood Repository at Brigham and Women’s Hospital – collects, stores, and provides quality analysis for tissue samples from mesothelioma patients treated at Brigham and Women’s Hospital and Dana-Farber Cancer Institute. The samples are used to further ongoing mesothelioma-related studies and for archival storage and future research. With more than 1400 samples representing more than 200 patients, it is the largest such tissue bank in the world. Back to the Top
8. Researchers Probe Role of Asbestos in Development of Mesothelioma
Although asbestos is the only substance consistently associated with the development of mesothelioma, many questions remain as to the mechanisms by which it causes malignancy and whether there are, in fact, other currently unrecognized substances that also contribute to mesothelioma.
John J. Godleski, MD, head of pulmonary pathology at Brigham and Women’s Hospital and a member of the International Mesothelioma Program (IMP) research team, said, “Our research focuses on concentrations of fiber types in patients with mesothelioma for the purpose of defining the fiber types that contribute to the disease, and how their presence in the body begins the cascade that results in the development of mesothelioma.”
Godleski and his colleague Rick Rogers, PhD, director of the Biomedical Imaging Laboratory at Harvard School of Public Health, have recently presented results of preclinical studies demonstrating the use of innovative techniques – confocal microscopy, in vivo (in living tissue) chemiluminescence, and scanning electron microscopy – to elucidate the mechanisms of translocation, the process by which asbestos fibers deposited in the small airways of the lung are transported to the parietal pleura (lining of the lung) and, less frequently, to the visceral pleura (lining of the abdomen) where mesothelioma develops.
Confocal microscopy is an optical imaging technique that creates cross-sectional “slices” of a tissue fragment and reconstructs them into a three-dimensional image that allows researchers to see where fibers reside in the tissue. This novel approach, pioneered by Rogers, uses confocal microscopy to see and quantify asbestos fiber distribution in specific locations within the lung and pleura.
In vivo chemiluminescence detects the burden of reactive oxygenation species (ROS) within a tissue sample. ROS are normal byproducts of oxygen metabolism that, at high levels, damage DNA and are implicated in the development of malignancy. In vivo chemiluminescence techniques can direct attention to the timing and location of this critical process.
Electron microscopy is used to identify fiber types.
Godleski and Rogers are poised to begin further studies using these techniques to document translocation pathways. According to Godleski, “Increased understanding of these pathways may potentially point to ways to disrupt the processes inherent in the development of mesothelioma. It may suggest ways by which we can slow down or change the translocation process, remove fibers from vulnerable areas such as the lymphatics, or manipulate the lymphatic flow.”
In addition to translocation studies, Godleski’s lab also studies the concentration and distribution of asbestos fibers in tissue from mesothelioma patients. Cells of the lung respond to asbestos by forming an iron coating that is readily detected by light microscopy. Godleski, Karl T. Kelsey, MD, Professor of Environmental Pathology at Brown University, and others have demonstrated that this information is prognostically significant, as patients with lower body burdens of asbestos have a survival advantage compared with those who have the highest burden.
The researchers also analyze patient tissue using electron microscopy, which reveals the type of fiber and suggests something about its source. They compare this information to information derived from detailed patient exposure assessments to learn more about what fibers are responsible for the development of mesothelioma, and potentially to identify new fiber types associated with the disease.
Godleski concluded, “Understanding the types and sources of asbestos and the transport of asbestos through the body are important because the incidence of mesothelioma continues to increase.” Asbestos use in the United States and worldwide peaked almost 30 years later than is generally appreciated – we have not yet seen the end of the latency period for asbestos exposure that occurred in the 1970s and 1980s – and worldwide use continues to be alarmingly high. In addition, new fibers are being introduced into the workplace and the environment that may prove to be dangerous. Carbon nanotubes, for example, are manmade substances widely used in nanotechnology, optics, and electronics. They have many of the properties that make asbestos attractive, but perhaps they also may have similarly dire public health consequences.
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9. Researchers Target Quality of Life Enhancement for Mesothelioma Patients
Physicians and scientists are making remarkable progress in discovering and refining treatments that extend and enhance the lives of mesothelioma patients, yet both the disease and the treatment remain grueling, and both can have devastating effects on the quality of life of patients and their loved ones.
A newly-reopened International Mesothelioma Program-funded study seeks to describe a quality-of-life “baseline” for mesothelioma patients, identify those factors that enhance and those that inhibit quality of life, and determine the impact of different treatment regimens (multimodality treatment, chemotherapy alone, other treatment, and supportive care) on patients’ quality of life.
Tamara Tilleman, MD/PhD, the principal investigator for the study hopes to enroll 400 patients that are treated at Brigham and Women’s Hospital and Dana-Farber Cancer Institute.
These patients will receive mailed questionnaires every three weeks for 36 weeks covering areas such as ongoing symptoms including pain, physical functioning, psychological and emotional concerns, and social supports. These prospectively collected data will form a database containing longitudinal profiles of pain and quality of life.
Using this database, researchers hope to obtain normative information by which to interpret the clinical significance of the quality-of-life scores.
A sub-study will involve mesothelioma survivors – those who have completed treatment and are disease-free after one year. Little is known about this group of patients, but as therapy improves and more patients survive longer, it is important to understand how survivors adjust to their disease, how their quality of life is affected, and what supports might be beneficial for them,” according to Alice Kornblith, PhD. “This aspect of our quality-of-life research program would begin to provide information on an unrecognized subset of the mesothelioma population.”
“Ultimately,” said Kornblith, “our goal is to identify areas where patients are experiencing quality-of-life-issues and target programs to address those needs. For example, if social support is lacking, the program might establish new support groups or facilitate use of the internet to connect patients to other mesothelioma patients. If inadequately treated pain or other symptoms are the most important issues, clinicians can enhance symptom management efforts.”
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