Newswise — Cambridge, MA. Mon. April 21, 2014 -- Researchers from MIT’s Koch Institute for Integrative Cancer Research, the Broad Institute of MIT and Harvard, and the Dana-Farber Cancer Institute have published a study in Nature Biotechnology demonstrating that new experimental and analytical techniques for studying rare tumor cells in the bloodstream can provide a minimally invasive window into the genetics of metastatic prostate cancer. Bringing together oncologists, engineers, and computational biologists, the team describes proof-of-concept work demonstrating how to accurately map mutations in the DNA of rare circulating tumor cells (CTCs). The work sets the foundation for future comprehensive surveys of the genomics of CTCs applied to other cancer types and across large numbers of samples.
“From a pragmatic standpoint, having the resolution to identify individual point mutations in tumor DNA for which targeted therapies already exist or are in development is critical to be able to make decisions on which treatment may be best for an individual patient. The application of this process to samples from prostate cancer patients demonstrates that information about single-nucleotide variants in individual CTCs can be used to understand the genomics of the underlying cancer,” explains Koch Institute faculty member J. Christopher Love, co-senior author of this work, who is also professor of chemical engineering at MIT and a Broad associate member.
“The ability to use blood biopsies to monitor prostate cancer would be paradigm changing,” says Jesse Boehm, assistant director of the Broad Institute’s Cancer Program and a co-senior author of the study. “We built an interdisciplinary team among the Dana-Farber, MIT’s Koch Institute, and the Broad Institute, to work together to overcome some of the biggest challenges. The ultimate result establishes a platform to find and sequence CTCs accurately that we hope will power the cancer research community around the world.”
The co-first authors of the paper represent several areas of expertise: Jens G. Lohr is an associated scientist at the Broad Institute in the Golub Laboratory and a medical oncologist at the Dana-Farber Cancer Institute; Viktor Adalsteinsson is a graduate student in the Love Laboratory at the Koch Institute and in the department of chemical engineering at MIT; and Kristian Cibulskis is an assistant director of informatics for the Cancer Genome Analysis group led by Gad Getz at the Broad Institute.
Lohr, who also treats patients at Dana-Farber, hopes that the work provides a step toward making genomic analysis of blood biopsies possible in the clinic. “We are providing a framework to determine mutations in the entire coding part of the genome of circulating tumor cells,” Lohr says. “This can be done with just a few single cells from the blood of cancer patients."
For Adalsteinsson, who lost his mother, Eve, in 2011 to a 15-year battle with stage IV, metastatic breast cancer, this is an important milestone of a long-standing commitment to conquering cancer. “I chose to dedicate my life to cancer research. While studying chemical engineering at Penn State, I worked at Genentech, Regeneron, Sanofi Pasteur, and more. I read the literature and thought of ways to save her life,” Adalsteinsson explains. “When I came to MIT and saw Professor Chris Love present at our department seminar, I immediately knew I wanted to work for him.”
Through a collaboration with the Broad Institute’s Genomics Platform and with cancer researchers at the Broad Institute and Dana-Farber Cancer Institute, Adalsteinsson and the rest of the team in Love’s laboratory at the Koch Institute formed a multi-institutional collaboration to overcome the barriers to sequencing circulating tumor cells. “This is a wonderful example of collaboration across disciplines and across institutions. Bringing together powerful technologies, here represented by MIT’s Koch Institute and the Broad Institute, with the clinical resources of the Dana-Farber Cancer Institute, has led to critical new insights into cancer biology,” says Koch Institute Director and David H. Koch Professor of Biology Tyler Jacks.
A systematic approach to sequencing CTCs
CTCs, cells that dislodge from primary tumors or metastatic sites and enter the bloodstream, are seen in a number of cancers and are thought to contribute to cancer dissemination and metastatic disease. The number of CTCs in circulation, approximately one in a million to one in 10 million cells, varies by disease, which may reflect our still incomplete understanding of the markers that signify these populations of cells in blood. In the clinic, CTCs have been used to determine prognosis of disease, predominantly by counting the number of tumor cells present in circulation. Although CTCs represent an interesting look into the disease itself because they provide a snapshot of its current stage, much of their biology and how they may reflect on the disease itself have not been well characterized.
The drop in the cost of genomic sequencing as well as advances in tools for amplifying the amount of genetic material of a single cell, i.e., making copies of its genome to sequence it comprehensively, have resulted in great advancements in technologies and approaches for sequencing single cells. Yet, the low number of CTCs in circulation and the noise associated with errors in the copy of the genetic material and with the sequencing technologies themselves, have historically made it difficult to sequence single-nucleotide variants in individual CTCs. The newly published study offers a set of experimental and analytical protocols to achieve the necessary level of sensitivity. “It is exciting to be able to accurately infer mutations in a tumor from only a few cancer cells detected in the blood,” says Gad Getz, a co-senior author of the study and director of the Broad Institute's Cancer Genome Computational Analysis group. Getz is also the director of the Bioinformatics Program at Massachusetts General Hospital Cancer Center and the department of pathology.
The strategy relies on a two-step enrichment to isolate individual CTCs followed by some advances in the ability to sequence and identify the variants in the genomes of these CTCs. The first step of the isolation process used for this proof-of-concept work relied on the use of the MagSweeper technology, developed by Stanford University and provided to the collaboration by the sequencing company Illumina. Using the MagSweeper, magnetic beads were used to achieve about a 10,000-fold enrichment in the number of CTCs in blood. The beads used in this study targeted a marker called epithelial cell adhesion molecule (EpCAM) that is expressed on the surface of tumor cells. But, a second enrichment step is necessary to purify the rare CTCs such that their genomes could be sequenced.
For that, the team used a technology that the Love Laboratory previously developed for isolating individual cells in arrays of sub-nanoliter wells. This allows them to image the cells, use surface-expressed markers to verify that they are in fact CTCs, and recover those individual cells in isolation to prepare 10-30 individual CTCs from the same patient and prepare their DNA for sequencing. Sequencing DNA from individual cells is extremely error-prone, so the team focused on DNA mutations that were seen in at least three independent CTCs. This “census” based method for determining which mutations were real was the key to unlock the method’s true power.
“In a typical experiment we may isolate 10 cells, and then look for variants that are present in at least three of those members. This approach allows us to call mutations that are shared among multiple individual cells,” says Love.
By design, the process is very modular and can easily accommodate new CTC isolation or identification technologies, whole-genome amplification methods, and sequencing platforms as they develop. The team plans to continue optimizing cost-effective strategies for single-cell sequencing not only of CTCs but also of biopsy materials from primary or metastatic lesions.
A window into the mutational landscape of metastatic prostate cancer
To show that the genomes of circulating tumor cells present in blood biopsies were, indeed, representative of a patient’s prostate tumor, the team compared the mutations from the CTCs with the mutations in the primary prostate cancer as well as a lymph node metastasis. Excitingly, 90% of the mutations that were ubiquitously detectable in all biopsies of the primary tumor as well as in the metastasis were also detected in the CTCs. Since prostate cancer often metastasizes to bone, sampling metastatic tumors is extremely challenging. The new blood biopsy sequencing approach may soon make it possible to perform clinical cancer genomics for prostate and other tumors much more readily. "This paper demonstrates the feasibility and rigorous methodology required for whole exome sequencing of single circulating tumor cells. It is a tour de force that paves the way for comprehensive genotyping of the different cancer cells shed during disease progression. By providing a systematic process for revealing changes that occur in cancer cells as they spread, it offers a way to both advance our understanding of cancer biology and ultimately guide cancer treatment for individual patients in real-time," says Stefanie Jeffrey, MD, the John and Marva Warnock Professor and chief of surgical oncology research at Stanford University School of Medicine.
The group plans to further investigate the relationship between the genomes of these CTCs and the diversity of metastatic sites in many more patients, and whether the variants that they see in the circulating population always reflect the overall disease of the subject. “The extent to which these CTCs represent every metastatic lesion is not yet known, but our results suggest that we can use this population of CTCs to understand the underlying genomics of the cancer of the individual when there is metastatic disease that may be otherwise inaccessible,” Love points out.
The research team is now beginning to also use this approach to undertake longitudinal studies to monitor how CTCs from patients who are undergoing treatment change over time. “If there are changes in the genomes that are evolving under the pressure of a particular drug, we will be able to identify them in the CTCs, which could ultimately lead to information about mechanisms of resistance to therapies and new target identification, as well as help guide treatment choices.”
For Adalsteinsson, the impact of using these populations of cells in ways that can improve patient care and outcomes goes far beyond a technical and clinical achievement. “My mother passed away on August 21st of 2011. Two days after, I went back to the lab to fulfill my promise of finding a cure for so many others. I believe we are making great progress,” he says.
This work was supported in part by the Koch Institute Support (core) grant from the National Cancer Institute, a TRANSCEND grant from Janssen Pharmaceuticals, Inc., and the Howard Hughes Medical Institute (Dr. Todd Golub).
About the Broad Institute of MIT and HarvardThe Eli and Edythe L. Broad Institute of MIT and Harvard was launched in 2004 to empower this generation of creative scientists to transform medicine. The Broad Institute seeks to describe all the molecular components of life and their connections; discover the molecular basis of major human diseases; develop effective new approaches to diagnostics and therapeutics; and disseminate discoveries, tools, methods, and data openly to the entire scientific community.
Founded by MIT, Harvard, and its affiliated hospitals, and the visionary Los Angeles philanthropists Eli and Edythe L. Broad, the Broad Institute includes faculty, professional staff and students from throughout the MIT and Harvard biomedical research communities and beyond, with collaborations spanning over a hundred private and public institutions in more than 40 countries worldwide. For further information about the Broad Institute, go to http://www.broadinstitute.org. About the Koch Institute for Integrative Cancer ResearchThe Koch Institute for Integrative Cancer Research, a National Cancer Institute (NCI)-designated Cancer Center, is a state-of-the-art cancer research facility as well as the hub of cancer research on the MIT campus. Completed in 2010, the Koch Institute building allows for the physical co-localization of faculty members from the Department of Biology (formerly in the MIT Center for Cancer Research) with faculty members drawn from a variety of departments in the MIT School of Engineering. The Koch Institute faculty also includes many members located in other research buildings at MIT, including the Whitehead and Broad Institutes. The Koch Institute brings together biologists and chemists along with biological, chemical, mechanical, and materials science engineers, computer scientists, clinicians, and others, to bring fresh perspectives and an interdisciplinary approach to advancing the fight against cancer. This multi-faceted group of investigators is at the core of the Koch Institute’s mission to develop new insights into cancer, as well as new tools and technologies to better treat, diagnose, and prevent the disease. For further information about MIT’s Koch Institute visit to http://ki.mit.edu.
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Nature Biotechnology, April-2014, in press