QNAS
QNAS
QnAs with Stephen Quake Prashant Nair, Science Writer
Stephen Quake’s foray into molecular diagnosis began as a discomfiting personal experience. As he watched his pregnant wife undergo invasive diagnostic tests before the birth of his children, Quake came to grips with the startling risks posed by the procedures. Before long, he devised an ingenious workaround: a method to detect abnormal numbers of chromosomes in fetuses by counting and sequencing cell-free DNA molecules circulating in the maternal bloodstream. Reported in a 2008 PNAS article (1), the method broke new ground in the field of noninvasive prenatal diagnosis and spurred the development of commercial tests now used in clinics worldwide. (In related work, molecular biologist Dennis Lo, at the Chinese University of Hong Kong, demonstrated in the late 1990s that extra copies of chromosome 21 can be detected in the bloodstream of mothers carrying fetuses afflicted with Down’s syndrome.) Quake, a bioengineer at Stanford University and a member of the National Academy of Sciences, has since ventured into a diverse array of fields in biology. His pursuits stem from his basic training in biophysics and have converged in a raft of clinical applications, including the genomic analysis of cancer and transplant rejection. “It’s been rewarding to see how one can apply simple ideas about measurement from physics to a range of biological problems,” he says. So it is only fitting that the National Academy of Sciences bestowed on him the 2016 Raymond and Beverly Sackler Prize in Convergence Research. PNAS spoke to Quake to mark the occasion. PNAS: You have used single-cell gene sequencing methods to unearth insights into cancer progression and treatment. In a 2014 PNAS article (2), for example, you described how this approach can be used to trace the development of acute lymphoblastic leukemia [ALL] in children. Can you explain the insights you gleaned? Quake: In that work we analyzed the clonal structure of human tumors, particularly for children with ALL, a blood cancer. We were able to perform genome analysis on single cells from bone marrow samples from these patients and work out the natural history of their cancers. This gave us insight into the different clones within a given patient’s tumor that were competing with one another for dominance, and this is key to cancer drug resistance. Typically, cancer chemotherapy drugs kill the dominant clones within tumors, but resistant clones often take their place. With the ability to study individual clones at the single-cell level, we can begin to develop ways to attack all of the clones within a tumor and perhaps
www.pnas.org/cgi/doi/10.1073/pnas.1610547113
Stephen Quake. Image courtesy of Stanford Engineering.
suppress the emergence of resistance. The clinical fellow who led the work is now extending it to a broad range of tumors and trying to unravel how tumors evolve over time. PNAS: Years after their initial demonstration, exome and whole-genome sequencing methods have begun to play diagnostic and prognostic roles in the clinic, at least for some cancers and at some major medical centers in the United States. What kind of clinical role do you envision for single-cell genomics? Quake: I can imagine a number of clinical scenarios. Liquid tumors have certain inherent advantages for singlecell genomics; we can access the cells more easily than in solid tumors. With the explosion of interest in cellular therapies for cancer, such as CAR-T [chimeric antigen receptor T-cell] immunotherapies, single-cell genomics is likely to play a key role—likely as a quality-control tool in the clinic—within the next 5 to 10 years, at least for cancer. I also expect that single-cell genomics will play an important role in dissecting the heterogeneity of solid tumors, perhaps for more nuanced interpretation of biopsies. PNAS: You have also turned your attention to the human brain. In June 2015, you described the use of single-cell RNA sequencing to illuminate the astounding cellular complexity of the human fetal and adult brain in a PNAS article (3). Along the way, you uncovered rather surprising insights about the brain.
PNAS | August 2, 2016 | vol. 113 | no. 31 | 8557–8558
Quake: That paper used single-cell genomics to demonstrate the phenomenal heterogeneity in the human brain, which has an enormous number of cell types. We were fortunate to strike up a collaboration with Stanford neuroscientist Ben Barres and neurosurgeon Melanie Hayden Gephart, who provided us [with] healthy human cortical tissues from people she was operating on. There were a number of fascinating insights from that work. For example, we found that the mouse is not a particularly good model for studying the human brain, especially due to differences in the types and relative frequencies of neuronal surface markers between mouse and human neurons. We also showed that a subset of adult human neurons was expressing MHC immune molecules on their surfaces, which was a complete surprise, given that the human brain had been largely thought to be immune-privileged. PNAS: You are perhaps best known among biologists for your pioneering work on noninvasive methods of molecular diagnosis. Your 2008 PNAS article (1) on diagnosing fetal Down’s syndrome by sequencing DNA from maternal blood eventually led to a handful of noninvasive prenatal diagnostic tests. Tell us how you were beckoned by destiny, as it were, to enter this area of biology. Quake: My interest in this area stemmed from being a parent and seeing invasive tests being done to my wife and unborn child. It was somewhat shocking to me that we had to risk the life of the baby to ask a diagnostic question. Our work on single-molecule biophysics and novel DNA sequencing methods led us to explore ways to count single molecules in clinically relevant settings, and this effort represents a convergence of different fields, including genomics and high-precision physics. The PNAS paper (1) demonstrated a noninvasive fetal diagnostic method for Down’s syndrome, and it made a compelling argument for retiring amniocentesis as a prenatal technique. The work was independently replicated, and really changed the way prenatal diagnosis was carried out for more than a million women worldwide. Today, several companies are offering different versions of the test. Furthermore, this led us into noninvasive molecular diagnosis for recipients of heart and lung transplants, as well as patients with mysterious infectious diseases associated with uncharacterized microbes. The applications that have emerged are in various stages of commercialization. PNAS: Tell us more about the use of noninvasive methods for detecting rejection of heart and lung grafts in transplant recipients. Are these tests commercially available?
Quake: One day I got a call from cardiologist Hannah Valentine, and she described the problems they confronted in determining whether heart transplant patients were facing immune rejection. To test whether transplanted organs are under attack by the recipients’ immune systems, doctors generally rely on biopsies, which are invasive and can be painful. We developed a blood test that would replace the biopsies by simply measuring the cell-free DNA, from the transplanted organ, that was circulating in the recipient’s bloodstream. A commercial version of the test for heart transplant patients is being currently offered by CareDx, a company in which I am a consultant. PNAS: Liquid biopsies are slowly gaining prominence as diagnostic and prognostic tools for cancer: a spate of studies has proven the principle, and some companies are already offering tests to detect and monitor cancer. Have you applied your methods to the cancer arena? Quake: Cancer is a challenging disease to diagnose, particularly because of its heterogeneity and variability. At the risk of oversimplifying, every pregnancy is more or less the same, but every cancer is different. With cancer, there are any number of technical challenges, such as the amount of circulating, cell-free DNA that is detectable, depending on the tumor type, the clinical relevance of the window in which the tumor DNA can be detected, etc., So we have purposely stayed away from cancer, especially because a number of highly capable research groups and companies are working diligently in that space. PNAS: You have also used noninvasive methods to probe the health of various tissues in the human body. Can you explain how this work differs from your other pursuits in noninvasive diagnosis? Quake: For the applications I have discussed so far— cancer, transplants, prenatal diagnostics—the methods rely on detecting foreign genomes in the body [for cancer, the genomes are not foreign but mutant]. But for a number of diseases, we simply want to measure the state of health in a given tissue. For this we turned to measuring circulating RNA as a marker of tissue-specific gene expression. In a 2014 PNAS paper (4) we showed, for example, that we can measure transcripts from dying neurons in the blood of Alzheimer’s patients. A company called Molecular Stethoscope is using the method to commercialize tests for neurodegenerative, autoimmune, inflammatory, and coronary artery diseases, to name a few examples.
1 Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR (2008) Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci USA 105(42):16266–16271. 2 Gawad C, Koh W, Quake SR (2014) Dissecting the clonal origins of childhood acute lymphoblastic leukemia by single-cell genomics. Proc Natl Acad Sci USA 111(50):17947–17952. 3 Darmanis S, et al. (2015) A survey of human brain transcriptome diversity at the single cell level. Proc Natl Acad Sci USA 112(23):7285–7290. 4 Koh W, et al. (2014) Noninvasive in vivo monitoring of tissue-specific global gene expression in humans. Proc Natl Acad Sci USA 111(20): 7361–7366.
8558 | www.pnas.org/cgi/doi/10.1073/pnas.1610547113
Nair
QNAS
QnAs with Stephen Quake Prashant Nair, Science Writer
Stephen Quake’s foray into molecular diagnosis began as a discomfiting personal experience. As he watched his pregnant wife undergo invasive diagnostic tests before the birth of his children, Quake came to grips with the startling risks posed by the procedures. Before long, he devised an ingenious workaround: a method to detect abnormal numbers of chromosomes in fetuses by counting and sequencing cell-free DNA molecules circulating in the maternal bloodstream. Reported in a 2008 PNAS article (1), the method broke new ground in the field of noninvasive prenatal diagnosis and spurred the development of commercial tests now used in clinics worldwide. (In related work, molecular biologist Dennis Lo, at the Chinese University of Hong Kong, demonstrated in the late 1990s that extra copies of chromosome 21 can be detected in the bloodstream of mothers carrying fetuses afflicted with Down’s syndrome.) Quake, a bioengineer at Stanford University and a member of the National Academy of Sciences, has since ventured into a diverse array of fields in biology. His pursuits stem from his basic training in biophysics and have converged in a raft of clinical applications, including the genomic analysis of cancer and transplant rejection. “It’s been rewarding to see how one can apply simple ideas about measurement from physics to a range of biological problems,” he says. So it is only fitting that the National Academy of Sciences bestowed on him the 2016 Raymond and Beverly Sackler Prize in Convergence Research. PNAS spoke to Quake to mark the occasion. PNAS: You have used single-cell gene sequencing methods to unearth insights into cancer progression and treatment. In a 2014 PNAS article (2), for example, you described how this approach can be used to trace the development of acute lymphoblastic leukemia [ALL] in children. Can you explain the insights you gleaned? Quake: In that work we analyzed the clonal structure of human tumors, particularly for children with ALL, a blood cancer. We were able to perform genome analysis on single cells from bone marrow samples from these patients and work out the natural history of their cancers. This gave us insight into the different clones within a given patient’s tumor that were competing with one another for dominance, and this is key to cancer drug resistance. Typically, cancer chemotherapy drugs kill the dominant clones within tumors, but resistant clones often take their place. With the ability to study individual clones at the single-cell level, we can begin to develop ways to attack all of the clones within a tumor and perhaps
www.pnas.org/cgi/doi/10.1073/pnas.1610547113
Stephen Quake. Image courtesy of Stanford Engineering.
suppress the emergence of resistance. The clinical fellow who led the work is now extending it to a broad range of tumors and trying to unravel how tumors evolve over time. PNAS: Years after their initial demonstration, exome and whole-genome sequencing methods have begun to play diagnostic and prognostic roles in the clinic, at least for some cancers and at some major medical centers in the United States. What kind of clinical role do you envision for single-cell genomics? Quake: I can imagine a number of clinical scenarios. Liquid tumors have certain inherent advantages for singlecell genomics; we can access the cells more easily than in solid tumors. With the explosion of interest in cellular therapies for cancer, such as CAR-T [chimeric antigen receptor T-cell] immunotherapies, single-cell genomics is likely to play a key role—likely as a quality-control tool in the clinic—within the next 5 to 10 years, at least for cancer. I also expect that single-cell genomics will play an important role in dissecting the heterogeneity of solid tumors, perhaps for more nuanced interpretation of biopsies. PNAS: You have also turned your attention to the human brain. In June 2015, you described the use of single-cell RNA sequencing to illuminate the astounding cellular complexity of the human fetal and adult brain in a PNAS article (3). Along the way, you uncovered rather surprising insights about the brain.
PNAS | August 2, 2016 | vol. 113 | no. 31 | 8557–8558
Quake: That paper used single-cell genomics to demonstrate the phenomenal heterogeneity in the human brain, which has an enormous number of cell types. We were fortunate to strike up a collaboration with Stanford neuroscientist Ben Barres and neurosurgeon Melanie Hayden Gephart, who provided us [with] healthy human cortical tissues from people she was operating on. There were a number of fascinating insights from that work. For example, we found that the mouse is not a particularly good model for studying the human brain, especially due to differences in the types and relative frequencies of neuronal surface markers between mouse and human neurons. We also showed that a subset of adult human neurons was expressing MHC immune molecules on their surfaces, which was a complete surprise, given that the human brain had been largely thought to be immune-privileged. PNAS: You are perhaps best known among biologists for your pioneering work on noninvasive methods of molecular diagnosis. Your 2008 PNAS article (1) on diagnosing fetal Down’s syndrome by sequencing DNA from maternal blood eventually led to a handful of noninvasive prenatal diagnostic tests. Tell us how you were beckoned by destiny, as it were, to enter this area of biology. Quake: My interest in this area stemmed from being a parent and seeing invasive tests being done to my wife and unborn child. It was somewhat shocking to me that we had to risk the life of the baby to ask a diagnostic question. Our work on single-molecule biophysics and novel DNA sequencing methods led us to explore ways to count single molecules in clinically relevant settings, and this effort represents a convergence of different fields, including genomics and high-precision physics. The PNAS paper (1) demonstrated a noninvasive fetal diagnostic method for Down’s syndrome, and it made a compelling argument for retiring amniocentesis as a prenatal technique. The work was independently replicated, and really changed the way prenatal diagnosis was carried out for more than a million women worldwide. Today, several companies are offering different versions of the test. Furthermore, this led us into noninvasive molecular diagnosis for recipients of heart and lung transplants, as well as patients with mysterious infectious diseases associated with uncharacterized microbes. The applications that have emerged are in various stages of commercialization. PNAS: Tell us more about the use of noninvasive methods for detecting rejection of heart and lung grafts in transplant recipients. Are these tests commercially available?
Quake: One day I got a call from cardiologist Hannah Valentine, and she described the problems they confronted in determining whether heart transplant patients were facing immune rejection. To test whether transplanted organs are under attack by the recipients’ immune systems, doctors generally rely on biopsies, which are invasive and can be painful. We developed a blood test that would replace the biopsies by simply measuring the cell-free DNA, from the transplanted organ, that was circulating in the recipient’s bloodstream. A commercial version of the test for heart transplant patients is being currently offered by CareDx, a company in which I am a consultant. PNAS: Liquid biopsies are slowly gaining prominence as diagnostic and prognostic tools for cancer: a spate of studies has proven the principle, and some companies are already offering tests to detect and monitor cancer. Have you applied your methods to the cancer arena? Quake: Cancer is a challenging disease to diagnose, particularly because of its heterogeneity and variability. At the risk of oversimplifying, every pregnancy is more or less the same, but every cancer is different. With cancer, there are any number of technical challenges, such as the amount of circulating, cell-free DNA that is detectable, depending on the tumor type, the clinical relevance of the window in which the tumor DNA can be detected, etc., So we have purposely stayed away from cancer, especially because a number of highly capable research groups and companies are working diligently in that space. PNAS: You have also used noninvasive methods to probe the health of various tissues in the human body. Can you explain how this work differs from your other pursuits in noninvasive diagnosis? Quake: For the applications I have discussed so far— cancer, transplants, prenatal diagnostics—the methods rely on detecting foreign genomes in the body [for cancer, the genomes are not foreign but mutant]. But for a number of diseases, we simply want to measure the state of health in a given tissue. For this we turned to measuring circulating RNA as a marker of tissue-specific gene expression. In a 2014 PNAS paper (4) we showed, for example, that we can measure transcripts from dying neurons in the blood of Alzheimer’s patients. A company called Molecular Stethoscope is using the method to commercialize tests for neurodegenerative, autoimmune, inflammatory, and coronary artery diseases, to name a few examples.
1 Fan HC, Blumenfeld YJ, Chitkara U, Hudgins L, Quake SR (2008) Noninvasive diagnosis of fetal aneuploidy by shotgun sequencing DNA from maternal blood. Proc Natl Acad Sci USA 105(42):16266–16271. 2 Gawad C, Koh W, Quake SR (2014) Dissecting the clonal origins of childhood acute lymphoblastic leukemia by single-cell genomics. Proc Natl Acad Sci USA 111(50):17947–17952. 3 Darmanis S, et al. (2015) A survey of human brain transcriptome diversity at the single cell level. Proc Natl Acad Sci USA 112(23):7285–7290. 4 Koh W, et al. (2014) Noninvasive in vivo monitoring of tissue-specific global gene expression in humans. Proc Natl Acad Sci USA 111(20): 7361–7366.
8558 | www.pnas.org/cgi/doi/10.1073/pnas.1610547113
Nair
