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Cervical Cancer Clues

Cervical Cancer Clues

Genomic study points to potential therapeutics, highlights HPV

By VERONICA MEADE-KELLY
Thu, 2014-01-02

Squamous cell carcinoma in the cervix. Image: National Cancer Institute

Researchers from the Boston area, Mexico and Norway have completed a comprehensive genomic analysis of cervical cancer in two patient populations. The study identified recurrent genetic mutations not previously found in cervical cancer, including at least one for which targeted treatments have been approved for other forms of cancer. The findings also shed light on the role human papillomavirus (HPV) plays in the development of cervical cancer.

The study, which appears online in Nature, addresses a public health concern of global significance. Cervical cancer is the second most common cancer in women and is responsible for approximately 10 percent of cancer deaths in women—particularly in developing countries where screening methods are not readily accessible. Almost all cases of the disease are caused by exposure to HPV and it is expected that vaccination efforts targeting HPV will decrease cervical cancer cases over time. In the meantime, however, the disease remains a significant threat to women’s health.

“Cancer is a disease that affects the whole world, and one question always arises: Is a given cancer type similar or different across populations?” explained Matthew Meyerson, one of the paper’s co-senior authors. Meyerson is an HMS professor of pathology and medical oncology at Dana-Farber Cancer Institute and a senior associate member of the Broad Institute. “While we don’t have the complete answer yet in this case, what we are seeing is that, in two different populations, the causes of cervical cancer are similar and, fundamentally, in both cases it comes down to HPV-genome interaction.”

To investigate the genomic underpinnings of the disease, the team performed whole-exome sequencing, which examines the genetic code in the protein-coding regions of the genome, on samples from 115 cervical cancer patients from Norway and Mexico. In some cases, the researchers also conducted whole-genome sequencing (analyzing the genetic code across the entire genome) or transcriptome sequencing (focusing on gene expression). In each case, the researchers compared genomic data derived from cervical cancer tumors with genomic data from healthy tissue from the same individual to determine what may have gone wrong—or mutated—in the genome to allow the cancer to develop. The mutations identified in tumors but not in healthy tissues from the same individuals are referred to as somatic mutations.

The study benefited from the international collaboration of scientists from research institutes across the globe and was made possible by the Slim Initiative for Genomic Medicine in the Americas (SIGMA), which promotes the study of genomic medicine in the service of global health.

“Low- and middle-income countries suffer the largest burden of cancer in the world,” said co-author Jorge Melendez of the National Institute of Genomic Medicine in Mexico City. “Nevertheless, only 5 percent of all the global resources dedicated to this group of diseases are allocated to them. Initiatives that promote joint efforts with developing countries will help to advance not only the knowledge of the shared and distinct biological aspects of cancer diseases, but also highlight local action items to impact public health.”

The cooperation of teams from the U.S., Mexico and Norway was essential in order to sequence samples from a diverse pool of cervical cancer patients.

“Without this sort of international collaboration, the genomic view of a disease can be limited. By analyzing genomic data from diverse populations, we can discover patterns of disease progression in context of the full range of human genetic variation,” said co-senior author Helga Salvesen. Salvesen, a professor of clinical medicine at the University of Bergen, Norway, was a visiting scientist at the Broad Institute when the study was conducted. 

The study identified 13 mutations that occurred frequently enough across the samples to be considered significant in cervical cancer. Eight of these mutations had not been linked to the disease previously, and two had not previously been seen in any cancer type.

Among the most notable findings were somatic point mutations in the gene ERBB2, which was found in a small but significant subset of the tumors. Mutations in this gene, which is also known as HER-2, had not been previously linked to cervical cancer, but it is a known oncogene common in breast cancer. Treatments exist that target the gene.

“This suggests that a subset of cervical cancer patients could be candidates for clinical trials involving ERBB2 inhibitors, which are available and FDA approved,” explained Akinyemi Ojesina, an HMS postdoctoral fellow in Meyerson’s lab at the Broad Institute and Dana-Farber. Ojesina served as a co-first author of the paper along with Lee Lichtenstein of the Broad’s Cancer Genome Analysis Group. “It is an exciting finding that could be translatable to the clinic.”

The team also identified a novel mutation in the gene MAPK1. MAPK1 is one of the final steps in the MAP kinase signaling pathway—a network of interconnected genes that play a role in cell growth regulation. Mutations in other genes in the pathway have been known to drive cancer, but this is the first time that MAPK1 itself has been found to be mutated. The finding opens up the possibility that MAPK1, like other genes in the MAP kinase signaling pathway, may be a viable therapeutic target.

Another key finding was the prevalence of mutations in genes affecting the immune system, in particular those in the gene HLA-A, which helps the body distinguish its own proteins from foreign invaders. Mutations in HLA-A were previously found to drive squamous cell lung cancer. In this study, another gene in the same complex—HLA-B—was found to be commonly mutated in cervical squamous cell carcinoma. This suggests that disruptions to the immune system may play a bigger role in cancer progression than was previously realized.

Finally, transcriptome sequencing, which allowed the team to analyze gene expression—how and when genes are activated across the genome—enabled the researchers to learn more about how HPV is driving cervical cancer.

It has long been known that exposure to HPV is a primary risk factor for cervical cancer. Once an individual is exposed, the immune system often clears out the infection, but in cases in which the virus lingers, it can integrate itself into the human genome. This study looked at where in the genome HPV inserted itself. The scientists found that HPV integration sites were associated with higher levels of gene expression and were often amplified, resulting in many copies of those sections of the genome. This connection between HPV integration and gene expression suggests that the virus may be driving cancer by promoting and elevating the activity of mutated genes.

“Our findings further elucidate the key role HPV is playing in the development of cervical cancer, which in turn emphasizes the importance of combating the disease by vaccinating against HPV,” Meyerson said.

In addition to the evidence supporting vaccination as a means of prevention, the researchers say the study bears important clinical implications for targeted therapeutics.

“In metastatic cervical cancer, more effective systemic therapy is urgently needed,” Salvesen explained. “So far, our knowledge regarding genetic alterations as potential targets for therapeutics has been limited, and no targeted therapeutics are yet in routine clinical use. The present study—in particular the findings related to ERBB2—thus represents a unique and comprehensive new tool to guide clinical trial design in the future.”

“The outstanding findings of our successful collaborative international research is giving us, here in Mexico, very powerful arguments in favor of the benefit of speeding up the adoption of molecular methods for screening HPV, followed by colposcopy to detect cancer at its earliest phases when it is curable; not to mention the possibility of new targeted therapies that our discoveries open up,” said Hugo Barrera, a professor of biochemistry and molecular medicine at the School of Medicine and University Hospital of the University of Nuevo Leon in Monterrey, Mexico, who, with Melendez, coordinated the Mexican team.

“It is hoped that, as we combine vaccination strategies and novel targeted therapies, we will be better able to combat the scourge that is cervical cancer,” Ojesina added.

This work was conducted as part of the Slim Initiative for Genomic Medicine in the Americas, a project funded by the Carlos Slim Foundation through the Health Institute. This work was also partially supported by the Rebecca Ridley Kry Fellowship of the Damon Runyon Cancer Research Foundation; MMRF Research Fellow Award; Helse Vest, Research Council of Norway, Norwegian Cancer Society and Harald Andersens legat; CONACyT grant SALUD-2008-C01-87625 and UANL PAICyT grant CS1038-1; and CONACyT grant 161619.

Adapted from a Broad Institute news release.

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A New, Reversible, Cause of Aging

A New—and Reversible—Cause of Aging

A naturally produced compound rewinds aspects of age-related demise in mice

By DAVID CAMERON
Thu, 2013-12-19

Mitochondria, organelles on the right, interact with the cell's nucleus to ensure a healthy, functioning cell. Image by Ana GomesResearchers have discovered a cause of aging in mammals that may be reversible.

The essence of this finding is a series of molecular events that enable communication inside cells between the nucleus and mitochondria. As communication breaks down, aging accelerates. By administering a molecule naturally produced by the human body, scientists restored the communication network in older mice. Subsequent tissue samples showed key biological hallmarks that were comparable to those of much younger animals.

“The aging process we discovered is like a married couple—when they are young, they communicate well, but over time, living in close quarters for many years, communication breaks down,” said Harvard Medical School Professor of Genetics David Sinclair, senior author on the study. “And just like with a couple, restoring communication solved the problem.”

This study was a joint project between Harvard Medical School, the National Institute on Aging, and the University of New South Wales, Sydney, Australia, where Sinclair also holds a position.

The findings are published Dec. 19 in Cell.

Communication breakdown

Mitochondria are often referred to as the cell's "powerhouse," generating chemical energy to carry out essential biological functions. These self-contained organelles, which live inside our cells and house their own small genomes, have long been identified as key biological players in aging. As they become increasingly dysfunctional overtime, many age-related conditions such as Alzheimer’s disease and diabetes gradually set in.

Researchers have generally been skeptical of the idea that aging can be reversed, due mainly to the prevailing theory that age-related ills are the result of mutations in mitochondrial DNA—and mutations cannot be reversed.

Sinclair and his group have been studying the fundamental science of aging—which is broadly defined as the gradual decline in function with time—for many years, primarily focusing on a group of genes called sirtuins. Previous studies from his lab showed that one of these genes, SIRT1, was activated by the compound resveratrol, which is found in grapes, red wine and certain nuts.

Sirt1 protein, red, circles the cell's chromosomes, blue. Image by Ana GomesAna Gomes, a postdoctoral scientist in the Sinclair lab, had been studying mice in which this SIRT1 gene had been removed. While they accurately predicted that these mice would show signs of aging, including mitochondrial dysfunction, the researchers were surprised to find that most mitochondrial proteins coming from the cell’s nucleus were at normal levels; only those encoded by the mitochondrial genome were reduced.

“This was at odds with what the literature suggested,” said Gomes.

As Gomes and her colleagues investigated potential causes for this, they discovered an intricate cascade of events that begins with a chemical called NAD and concludes with a key molecule that shuttles information and coordinates activities between the cell’s nuclear genome and the mitochondrial genome. Cells stay healthy as long as coordination between the genomes remains fluid. SIRT1’s role is intermediary, akin to a security guard; it assures that a meddlesome molecule called HIF-1 does not interfere with communication.

For reasons still unclear, as we age, levels of the initial chemical NAD decline. Without sufficient NAD, SIRT1 loses its ability to keep tabs on HIF-1. Levels of HIF-1 escalate and begin wreaking havoc on the otherwise smooth cross-genome communication. Over time, the research team found, this loss of communication reduces the cell's ability to make energy, and signs of aging and disease become apparent.

“This particular component of the aging process had never before been described,” said Gomes.

While the breakdown of this process causes a rapid decline in mitochondrial function, other signs of aging take longer to occur. Gomes found that by administering an endogenous compound that cells transform into NAD, she could repair the broken network and rapidly restore communication and mitochondrial function. If the compound was given early enough—prior to excessive mutation accumulation—within days, some aspects of the aging process could be reversed.

When Sirt1 loses its ability to monitor HIF-1, communication between mitochondria and the nucleus breaks down, and aging accelerates. Image by Ana Gomes

Cancer connection

Examining muscle from two-year-old mice that had been given the NAD-producing compound for just one week, the researchers looked for indicators of insulin resistance, inflammation and muscle wasting. In all three instances, tissue from the mice resembled that of six-month-old mice. In human years, this would be like a 60-year-old converting to a 20-year-old in these specific areas.

One particularly important aspect of this finding involvesHIF-1. More than just an intrusive molecule that foils communication, HIF-1 normally switches on when the body is deprived of oxygen. Otherwise, it remains silent. Cancer, however, is known to activate and hijack HIF-1. Researchers have been investigating the precise role HIF-1 plays in cancer growth.

“It’s certainly significant to find that a molecule that switches on in many cancers also switches on during aging,” said Gomes. “We're starting to see now that the physiology of cancer is in certain ways similar to the physiology of aging. Perhaps this can explain why the greatest risk of cancer is age.”

“There’s clearly much more work to be done here, but if these results stand, then certain aspects of aging may be reversible if caught early,” said Sinclair.

The researchers are now looking at the longer-term outcomes of the NAD-producing compound in mice and how it affects the mouse as a whole. They are also exploring whether the compound can be used to safely treat rare mitochondrial diseases or more common diseases such as Type 1 and Type 2 diabetes. Longer term, Sinclair plans to test if the compound will give mice a healthier, longer life.

The Sinclair lab is funded by the National Institute on Aging (NIA/NIH), the Glenn Foundation for Medical Research, the Juvenile Diabetes Research Foundation, the United Mitochondrial Disease Foundation and a gift from the Schulak family.

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Silencing Sudden Death

Silencing Sudden Death

Study targets genetics of hypertrophic cardiomyopathy

By STEPHANIE DUTCHEN
Thu, 2013-10-03

The heart of a mouse with untreated hypertrophic cardiomyopathy (left) has a thicker muscle wall and more scarring (blue) than one treated with RNAi (right). Image: Seidman lab

Hypertrophic cardiomyopathy (HCM)—a disease in which cardiac muscle thickens, weakening the heart—can be prevented from developing for several months in mice by reducing production of a mutant protein, according to a new study by researchers at Harvard Medical School.

The work takes a first step toward being able to treat or prevent the leading cause of sudden death in athletes and sudden heart-related death in people under 30 in the United States.

“There’s really no treatment for HCM right now. You can treat symptoms like chest pain or an arrhythmia, but that’s not getting at the fundamental problem,” said Christine Seidman, the Thomas W. Smith Professor of Medicine and Genetics at HMS and Brigham and Women's Hospital, a Howard Hughes Medical Investigator and senior author of the study. “While the application of this strategy is in the very early stages, it shows considerable promise.”

The results were published in Science on Oct. 3.

An estimated 1 in 500 Americans has HCM. Although many of them never develop symptoms, for others the disease can be severe or fatal.

More than 1,000 different mutations that can cause HCM have been identified across about 10 genes that make heart muscle proteins. People with HCM have one “good” copy and one “bad” copy of one of those genes.

Studying one of the mutations that causes particularly severe disease, Christine Seidman and Jonathan Seidman, Henrietta B. and Frederick H. Bugher Foundation Professor of Genetics at HMS, worked with research fellow Jianming Jiang and instructor Hiroko Wakimoto to target the analogous “bad” gene in mice while leaving the “good” gene alone.

The researchers created an RNA interference (RNAi) tool designed to home in on the single HCM-causing mutation and stop it from making its harmful protein. They packaged the RNAi inside a virus (a common RNAi delivery technique) and injected it into lab mice engineered to develop HCM. They compared the results to two untreated groups of mice: one with the same HCM mutation, and one without.

By suppressing the “bad” gene, the RNAi was able to reduce production of the mutant protein by about 28 percent. That was enough to prevent development of HCM manifestations—including ventricular wall overgrowth, cell disorganization and fibrosis (scarring)—for about six months, or one-quarter of the mice's lifespans.

"For all intents and purposes, the heart looked normal," said Christine Seidman. "Wonderfully, boringly normal."

The treatment successfully targeted heart cells in the mice without affecting other organs. Although it did not reverse any existing HCM damage, Jonathan Seidman noted that halting the progress of HCM would be a significant advance in itself.

"If somebody already had a certain amount of wall thickness and you prevent it from worsening, that would be a step forward to limit progressive symptoms and development of heart failure," he said.

In addition to its potential for informing HCM treatment in humans down the road, the initial findings could be relevant for a related genetic condition called dilated cardiomyopathy, where the heart becomes baggy and thin-walled and contracts too little instead of too much.

The researchers now plan to investigate whether they can continue to delay HCM in mice with booster shots, reverse disease damage or reduce HCM-related arrhythmias. They would like to study a larger animal model as well as explore whether younger mice respond better to therapy than older mice and if interventions aimed at specific areas of the heart could be as effective as treating the whole heart.

The team also intends to explore whether a collection of about 10 RNAis could be engineered to target common genetic variants that are tightly linked to HCM mutations, instead of having to develop 1,000 mutation-specific RNAis.

The Seidmans are founders of and own shares in MyoKardia, a biotechnology company developing small molecules that target the sarcomere (the cellular structure that contracts in heart muscle) for treatment of inherited cardiomyopathy.

This work was supported by grants from the National Institutes of Health/National Heart, Lung, and Blood Institute (U01HL09166 and R01HL084553) and the Howard Hughes Medical Institute.

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Glowing Proteins: More Than a Marker

Glowing Proteins: More Than a Marker

Researchers transform fluorescent proteins into a scaffold for manipulating genes

By ELIZABETH COONEY
Thu, 2013-08-15

GFP (green) can be used not only as a fluorescent reporter for labeling cells, but also as a scaffold to control synthetic biological activities, such as gene expression (red). Image: Jonathan Tang, with contributions by Tim Cherry.

Jonathan Tang had a problem. A graduate student studying neural circuitry in the retina, he wanted to do more than identify fluorescent cells that send signals to the brain. He sought to understand how these specialized cells called bipolar neurons develop and function in the eye’s retina. More than “Here they are,” he hoped to say, “Here’s what they do.”

Frustrated by the lack of available tools, he created his own. Tang, with Constance Cepko, HMS Bullard Professor of Genetics and Neuroscience, and other collaborators, transformed green fluorescent protein (GFP), a glowing biomarker borrowed from jellyfish, into a scaffold that can bring together protein fragments to enable gene manipulation, or other useful activities.

Their method, described Aug. 15 in Cell, enables scientists to probe how cells function not only in the retina, but also in other tissues. The tool also works with the promising technology called optogenetics, in which scientists use light to control individual cells.

To solve his problem, Tang took a step back to look more closely at GFP. The green glow produced by this jellyfish protein has become a workhorse of science, literally illuminating pathways and processes in lab dishes and living animal models since its Nobel Prize-winning discovery in 1961 and application in 1994. With it, scientists can observe molecular biology occurring in more than 1,500 transgenic GFP strains in mice and other organisms, many of which label unique populations of cell types.

An invaluable resource, GFP is nonetheless limited to tagging cells. If scientists want to control gene activity, particularly in the mouse, they turn to another workhorse molecule, Cre recombinase, an enzyme that can shuffle DNA and is primarily used in the mouse. Not nearly as many mouse strains have been made to express Cre as have GFP, so for Tang to learn not just where bipolar cells are in the retina, but also how they develop and connect to other cell types, additional transgenic mice would need to be generated and characterized.

Tang decided not to wait. After all, he had a PhD to complete.

“Jonathan is the star of the story,” Cepko said. “We would sit and talk about how we had all these GFP lines we couldn't use to derive functional information. He came up with this idea that worked fantastically well, namely, to use the GFP to control biological activity specifically in cells already tagged with GFP.”

To get there, Tang focused not so much on the green fluorescence in GFP as on its possible uses as a protein. By itself GFP doesn’t alter cell function—an asset as a biomarker—but perhaps it could be combined with other proteins that affect gene activity. In that sense, GFP and its partners could act as both biomarker and synthetic system for controlling genes.

Searching for proteins that would bind to GFP, Tang discovered that a group of scientists in Germany had identified molecules that could bind to GFP. Derived from camel antibodies prized for their simple structure and high affinity, these proteins retained their ability to bind to GFP after they were introduced into a cell. Tang realized that this property could be exploited to create an array of tools that could enable the GFP-binding proteins to do much more. He constructed a series of chimeric, or fusion, proteins, fusing protein domains that can control transcription to these GFP-binding proteins. When two such fusion proteins that can bind simultaneously to GFP are introduced into a cell, GFP brings them together, triggering the designed co-dependent activity of the fusion proteins.

The scientists tested their tool in the retina, using it to switch on a fluorescent reporter gene in one experiment and to knock out a gene in another. As a boost to those who study neural function, they could trigger expression of channelrhodopsin, a protein that allows neural circuits to be controlled with light, thereby linking expression of GFP to the powerful tools of optogenetics. 

The tool worked in tissue culture as well as in living mice and zebrafish, another animal model favored by investigators.

“We can now use this as a way to access genetically defined cell types to see what’s essential for development, behavior, neural circuit processing—or whatever one wants to study,” said Tang.

“We can use these methods to probe function and development in the retina, but whatever your tissue is, you should be able to use these methods to study it,” Cepko said. “You don’t have to make a transgenic mouse or even have a GFP strain. You may be able to find an endogenous protein, or maybe even an RNA, that’s specific to your interest and do exactly what we did to make our tool.”

The study authors were supported by the Leonard and Isabelle Goldenson Research Fellowship, a European Research Council grant, a Swiss-Hungarian grant, TREATRUSH, SEEBETTER and OPTONEURO grants from the European Union and the Howard Hughes Medical Institute.
 

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New Project in IBD Genetics

New Project in IBD Genetics

Broad Institute and Mass General announce major sequencing initiative in inflammatory bowel disease

Fri, 2013-08-02

Image: iStock

The Broad Institute and Massachusetts General Hospital are launching a new initiative to perform large-scale exome sequencing in inflammatory bowel disease (IBD), a diagnosis—including Crohn’s disease and ulcerative colitis—that faces considerable unmet therapeutic need.

The recent emergence of rapid, efficient genome-sequencing technologies and the compelling evidence for the role of genetics in these disorders have motivated the founding of a collaborative sequencing effort between the two institutions with ties to Harvard Medical School. The endeavor will be geared toward the discovery of high-impact genetic variants influencing IBD risk that can serve as guides to future therapeutic development and diagnostic tools.

The initiative will be directed by experienced IBD researchers from Mass General and the Broad Institute, but it also aims to develop an exome-sequencing network that will allow researchers to partner and collaborate worldwide. Two large pilot exome-sequencing projects are being launched immediately as part of this initiative based on established genetic study designs that heighten the discovery of rare, high-impact risk and protective variants. The exome is the small portion of the genome that translates DNA into proteins.

Early-onset pediatric IBD

A major focus of the work will surround full-exome sequencing of the earliest-onset childhood IBD cases. It has long been recognized in many diseases that penetrant genetic risk factors—mutations in people who have symptoms—are much more likely to be found in cases with unusually early onset. The Broad Institute has completed more than 50,000 exomes in the past two years. Its technical expertise in generating and processing such sequencing data, along with population-level genetic variation patterns from these experiments, will provide a foundation for obtaining the best outcomes in the interpretation of these cases. Samples with IBD onset before 6 years of age—and in some cases going up to age 10—collected by IBD researchers around the world are welcome for inclusion in this study.

Adult-onset IBD case-control study

Genetic mapping has provided dramatic insights into IBD pathogenesis in recent years, but a substantial amount of heritability—in particular the role of strong-acting rare mutations—is yet to be elucidated. To deliver those insights, IBD cases with an unusually low burden of known genetic risk factors (particularly emphasizing those with positive family history, or from isolated or enriched populations, or both) will be contrasted with population controls that have an extremely high burden of known IBD risk factors, enhancing discovery of critical protective variants that may provide the best clues for therapeutic development. This project will also be initiated as an international collaboration, particularly among researchers with sample sets with Immunochip genotyping that will enable the immediate genetic identification of these enriched target populations. Immunochip is a tool that samples 200,000 sites in the genome previously tied to autoimmune and inflammatory diseases.

The National Human Genome Research Institute Large-Scale Sequencing Program will support pilot work for this project in the next 12 months, with all generated data made publicly available to the research community through standard National Institutes of Health databases.

“Genetics offers a peek into the pathways that protect against—or predispose to—the development of IBD,” said Ramnik Xavier, HMS professor of medicine and chief of gastroenterology at Mass General and a senior associate member at the Broad Institute. “Contributing results to public resources will allow the data to be integrated by researchers from a variety of disciplines. In the current research environment, important advances in biology are often made by finding innovative ways to analyze ‘big data,’ and I believe this open-access exome-sequencing project will provide an example of how publicly available data can inspire significant progress in the field of IBD.”

“This project will enable IBD genetics to take further leaps forward toward a clear and therapeutically actionable understanding of the molecular causes of disease,” said Mark Daly, HMS associate professor of medicine and chief of the Analytic and Translational Genetics Unit at Mass General and a senior associate member at the Broad Institute. “It is extremely exciting to be able to launch this transformative initiative as an international collaboration with rapid and open data sharing.”

Researchers who are interested in collaborating on this initiative may contact Daly or Xavier for more information.

Adapted from a joint Mass General and Broad Institute news release.

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Cholera's Evolution

Cholera’s Evolution

Study identifies genomic regions associated with cholera susceptibility in Bangladesh

By SUE McGREEVEY
Tue, 2013-07-16

People in the Ganges River Delta of Bangladesh have been affected by cholera for centuries. Image: NASA World Wind screenshot via Wikimedia Commons

An international research team has used a novel approach to identify genetic factors that appear to influence susceptibility to cholera, a disease that affects from 3 to 5 million people each year and causes more than 100,000 deaths. The findings indicate the importance of pathways involved in regulating water loss in intestinal cells and highlight the innate immune system’s key role in the body’s response to the bacteria that causes cholera.

The work involved investigators from Harvard Medical School and Massachusetts General Hospital; the Broad Institute; and the International Center for Diarrhoeal Disease Research, Bangladesh.

“We sought to understand cholera by studying the genetics of a population that has been affected by the disease for centuries: people in the Ganges River Delta of Bangladesh,” said Regina LaRocque, an assistant professor of medicine at Mass General and a co-senior author of the report published in Science Translational Medicine. “Our findings are just a first step, but they demonstrate how combining ancient history with the current impact of an infectious disease can be a powerful way of identifying human genes that are important to disease outcome.”

People contract cholera by consuming water or food contaminated with the bacteria Vibrio cholerae, which releases a toxic protein upon reaching the small intestine. This toxin binds to the intestinal surface, causing severe diarrhea and sometimes death from dehydration. Cholera or cholera-like illnesses have been reported in the Ganges Delta for centuries, and most recent global outbreaks of the disease originated in that region.

A potential fingerprint of cholera’s genetic impact could be the relative rarity in the region of people with blood type O, which confers an increased risk of severe cholera symptoms. The persistence of cholera in the Ganges Delta would be expected to exert an evolutionary force on the population, since individuals with gene variants that reduce their susceptibility to the disease would be more likely to survive and pass those variants along to their children.

To search for genomic regions that affect cholera susceptibility, the team employed a new two-step approach. The first step, developed by the Broad team, used a method called Composite of Multiple Signals (CMS) to scan the genomes of 126 individuals from the Ganges Delta for patterns that signal a long-term increase in the prevalence of particular DNA segments, indicating the effects of natural selection. That scan identified 305 regions under selective pressure, many of which are involved in two important biologic functions. One is regulation of the passage of water through intestinal cells via structures called potassium channels and the other is a signaling pathway involved in both the innate immune system and the maintenance of the intestinal lining.

The second step directly tested the potential impact of these selected regions on cholera susceptibility by comparing the genomes of 105 cholera patients from the region with the genomes of 167 individuals who did not contract the disease, despite being exposed to it in their homes. That comparison found that the genomic region most strongly associated with cholera susceptibility in this population was one that the CMS scan indicated was under strong selection pressure. Genes in this region relate to an innate immune signaling pathway. LaRocque’s team had previously shown this pathway to be activated by exposure to cholera toxin, and the current study identified the potential involvement of several additional genes in that pathway

“Understanding the basic biology of a disease is fundamental to making clinically relevant advances in treatment,” said LaRocque. “Our laboratory is now working on further studies of the innate immune response to cholera, and we believe this work will be highly relevant to developing improved vaccines.”

Support for the study includes National Institutes of Health grants TW007409, AI058935, AI079198 and NIH Innovator Award DP2-OD006514-01; grants from the Howard Hughes Medical Institute, the American Cancer Society and the Packard Foundation; and an MGH Claflin Distinguished Scholar Award.

Adapted from a Mass General news release.

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After Tamoxifen

After Tamoxifen

Biomarker predicts likelihood of ER-positive breast cancer recurrence after treatment

By SUE McGREEVEY
Tue, 2013-07-16

Breast cancer cell. Image: National Cancer Institute

A biomarker reflecting expression levels of two genes in tumor tissue may be able to predict which women treated for estrogen-receptor-positive (ER positive) breast cancer should receive a second estrogen-blocking medication after completing tamoxifen treatment. In their report published in the Journal of the National Cancer Institute, HMS researchers at the Massachusetts General Hospital Cancer Center describe finding that the HOXB13/IL17BR ratio can indicate which women are at risk for cancer recurrence after tamoxifen and which are most likely to benefit from continuing treatment with the aromatase inhibitor letrozole.

“Most patients with early-stage, estrogen-receptor-positive breast cancer remain cancer-free after five years of tamoxifen treatment, but they remain at risk of recurrence for 15 years or longer after their initial treatment,” said Dennis Sgroi, HMS professor of pathology at Mass General and lead author of the report. “Our biomarker identifies the subgroup of patients who continue to be at risk of recurrence after tamoxifen treatment and who will benefit from extended therapy with letrozole, which should allow many women to avoid unnecessary extended treatment.”

Previous research by Sgroi’s team, in collaboration with investigators from bioTheranostics Inc., discovered that the ratio between levels of expression of two genes—HOXB13 and IL17BR—in tumor tissue predicted the risk of recurrence of ER-positive, lymph-node-negative breast cancer, whether or not the patient was treated with tamoxifen. The current study of patients from MA.17, the highly successful clinical trial of letrozole, was designed to go further. It would evaluate the usefulness of the HOXB13/IL17BR ratio for predicting which tamoxifen-treated patients remained at risk of recurrence, but it also sought to identify who could benefit from continued treatment with letrozole.

To answer those questions, the investigators analyzed primary tumor samples and patient data from the placebo-controlled MA.17 trial, which confirmed the ability of extended letrozole therapy to improve survival after the completion of tamoxifen treatment. Tissue samples were available from 83 patients whose tumors recurred during the study period (31 who had received letrozole and 52 in the placebo group) and 166 patients with no recurrence (91 who had received letrozole and 75 who got the placebo).

Analysis of the tumor samples revealed that a high HOXB13/IL17BR ratio—meaning the expression level of HOXB13 is greater than that of IL17BR—predicts an increased risk for tumor recurrence after tamoxifen therapy, but that elevated risk drops significantly if a patient receives letrozole.

“This discovery means that about 60 percent of women with the most common kind of breast cancer can be spared unnecessary treatment with the concomitant side effects and costs,” said Paul Goss, HMS professor of medicine, director of the Breast Cancer Research Program at the MGH Cancer Center and a co-author of the report. “But more important, the 40 percent of patients who are at risk of recurrence can now be identified as needing continued therapy with letrozole, and many will be spared death from breast cancer.” 

Goss and Sgroi noted that their findings need to be validated by additional studies before they can be put into clinical practice.

The study was supported by National Institute of Health grant R01-CA112021, Department of Defense Breast Cancer Research Program grant W81XWH-04-1-0606, and grants from the Avon Foundation, the Breast Cancer Foundation, the NCI SPORE in breast cancer at MGH, and Novartis.

Adapted from a Mass General news release.

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Off-Target Gene Editing

Off-Target Gene Editing

Unwanted mutations indicate need to improve precision of CRISPR-Cas RNA-guided nucleases

By SUE McGREEVEY
Tue, 2013-06-25

Image: iStock

In the past year a group of synthetic proteins called CRISPR-Cas RNA-guided nucleases (RGNs) have generated great excitement in the scientific community as gene-editing tools. Exploiting a method that some bacteria use to combat viruses and other pathogens, CRISPR-Cas RGNs can cut through DNA strands at specific sites, allowing new genetic material to be inserted. 

Now a team of HMS researchers at Massachusetts General Hospital has found a significant limitation to the method’s use: CRISPR-Cas RGNs produce unwanted DNA mutations at sites other than the desired target.

“We found that expression of CRISPR-Cas RGNs in human cells can have off-target effects that, surprisingly, can occur at sites with significant sequence differences from the targeted DNA site,” said J. Keith Joung, HMS associate professor of pathology at Mass General and associate chief of pathology, research in the Mass General Department of Pathology. He is co-senior author of the report published online in Nature Biotechnology. “RGNs continue to have tremendous advantages over other genome-editing technologies, but these findings have now focused our work on improving their precision.”

Consisting of a DNA-cutting enzyme called Cas9, coupled with a short, 20-nucleotide segment of RNA that matches the target DNA segment, CRISPR-Cas RGNs mimic the primitive immune systems of certain bacteria. When these microbes are infected by viruses or other organisms, they copy a segment of the invader’s genetic code and incorporate it into their DNA, passing it on to future bacterial generations. If the same pathogen is encountered in the future, the bacterial enzyme Cas9, guided by an RNA sequence that matches the copied DNA segment, inactivates the pathogen by cutting its DNA at the target site. 

About a year ago, scientists reported the first use of programmed CRISPR-Cas RGNs to target and cut specific DNA sites. Since then several research teams, including Joung’s, have successfully used CRISPR-Cas RGNs to make genomic changes in fruit flies, zebrafish, mice and in human cells—including induced pluripotent stem cells that have many of the characteristics of embryonic stem cells. The technology’s reliance on such a short RNA segment makes CRISPR-Cas RGNs much easier to use than other gene-editing tools called zinc finger nucleases (ZFNs) and transcription activator-like effector nucleases (TALENs). RGNs can also be programmed to introduce several genetic changes at the same time.

The possibility that CRISPR-Cas RGNs might cause additional, unwanted genetic changes has been largely unexplored, so Joung’s team set out to investigate the occurrence of “off-target” mutations in human cells expressing CRISPR-Cas RGNs. Since the interaction between the guiding RNA segment and the target DNA relies on only 20 nucleotides, they hypothesized that the RNA might also recognize DNA segments that differed from the target by a few nucleotides. 

Although previous studies had found that a single-nucleotide mismatch could prevent the action of some CRISPR-Cas RGNs, the MGH team’s experiments in human cell lines found multiple instances in which mismatches of as many as five nucleotides did not prevent cleavage of an off-target DNA segment. They also found that the rates of mutation at off-target sites could be as high as, or even higher than, those at the targeted site, something that has not been observed with off-target mutations associated with ZFNs or TALENs.

“Specificity is important both for research and especially for gene therapy,” George Church, the Robert Winthrop Professor of Genetics at HMS, said about Joung’s report. “This is the first paper to seriously address this topic. The next big question is how to reduce the off-target ratio to on-target.”

In January, Church reported in Science on research using the genome-editing tool. While he was not involved in the current study reported in Nature Biotechnology, Church and Joung are collaborators. Together with George Daley, HMS professor of biological chemistry and molecular pharmacology, and Kun Zhang, associate professor of bioengineering at the University of California at San Diego, they are co-principal investigators of a National Human Genome Research Institute Center for Excellence in Genomic Science.

Joung said RGNs remain valuable.

“Our results don’t mean that RGNs cannot be important research tools, but they do mean that researchers need to account for these potentially confounding effects in their experiments.  They also suggest that the existing RGN platform may not be ready for therapeutic applications,” said Joung. “We are now working on ways to reduce these off-target effects, along with methods to identify all potential off-target sites of any given RGN in human cells so that we can assess whether any second-generation RGN platforms that are developed will be actually more precise on a genome-wide scale. I am optimistic that we can further engineer this system to achieve greater specificity so that it might be used for therapy of human diseases.”

Support for the study includes National Institutes of Health (NIH) Director’s Pioneer Award DP1 GM105378; NIH grants R01 GM088040 and P50 HG005550, DARPA grant W911NF-11-2-0056, and the Jim and Ann Orr MGH Research Scholar Award.

Adapted from a Mass General news release.

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New MATH for Cancer

New MATH for Cancer

In head and neck tumors, genetic diversity predicts outcomes

By SUE McGREEVEY
Mon, 2013-05-20

Oral cancer squamous cell carcinoma, one of several types of head and neck cancer. Image: Wikimedia Commons

A new method of measuring the variety of genetic mutations found in cells within a tumor appears to predict treatment outcomes of patients with the most common type of head and neck cancer. In the May 20 issue of the journal Cancer, HMS investigators at Massachusetts General Hospital and Massachusetts Eye and Ear described how their way of measuring tumor heterogeneity was a better predictor of survival than are most traditional risk factors in a small group of patients with squamous cell carcinoma of the head and neck. 

“Our findings will eventually allow better matching of treatments to individual patients, based on this characteristic of their tumors,” said Edmund Mroz, HMS associate professor of physiology in the Department of Otology and Laryngology at Mass General’s Center for Cancer Research and lead author of the Cancer paper. “This method of measuring heterogeneity can be applied to most types of cancer, so our work should help researchers determine whether a similar relationship between heterogeneity and outcome occurs in other tumors.”

For decades investigators have hypothesized that tumors with a high degree of genetic heterogeneity—the result of different subgroups of cells undergoing different mutations at different DNA sites—would be more difficult to treat because particular subgroups might be more likely to survive a particular drug or radiation or to have spread before diagnosis.  While recent studies have identified specific genes and proteins that can confer treatment resistance in tumors, there previously has been no convenient way to measure tumor heterogeneity.

Working in the laboratory of James Rocco, who is the HMS Daniel Miller Associate Professor of Otology and Laryngology at Mass Eye and Ear, Mroz and his colleagues developed their new method by analyzing advanced gene-sequencing data to produce a value reflecting the genetic diversity within a tumor—not only the number of genetic mutations but how broadly particular mutations are shared within different subgroups of tumor cells. They first described this measure, called mutant-allele tumor heterogeneity (MATH), in the March 2013 issue of Oral Oncology.  But that paper was able to show only that patients with known factors predicting poor outcomes—including specific mutations in the TP53 gene or a lack of infection with the human papillomavirus (HPV)—were likely to have higher MATH values. 

In the current study, the investigators used MATH to analyze genetic data from the tumors of 74 patients with squamous cell head and neck carcinoma for whom they had complete treatment and outcome information. Not only did they find that higher MATH values were strongly associated with shorter overall survival—with each unit of increase reflecting a 5 percent increase in the risk of death—but that relationship was also seen within groups of patients already at risk for poor outcomes. For example, among patients with HPV-negative tumors, those with higher MATH values were less likely to survive than those with lower MATH values. Overall, MATH values were more strongly related to outcomes than were most previously identified risk factors. MATH values also improved outcome predictions based on all other risk factors the researchers examined.

The impact of MATH values on outcome appeared strongest among patients treated with chemotherapy, which may reflect a greater likelihood that highly heterogeneous tumors contain treatment-resistant cells, Mroz said. He also noted that what reduces the chance of survival appears to be the subgroups of cells with different mutations within a tumor, not the process of mutation itself. 

“If all the tumor cells have gone through the same series of mutations, a single treatment might still be able to kill all of them,” he said. “But if there are subgroups with different sets of mutations, one subgroup might be resistant to one type of treatment, while another subgroup might resist a different therapy.”

In addition to combining MATH values with clinical characteristics to better predict a patient’s chance of successful treatment, MATH could someday help determine treatment choice—directing the use of more-aggressive therapies against tumors with higher values, while allowing patients with lower values to receive less-intense standard treatment, Mroz noted. While MATH will probably be just as useful at predicting outcomes for other solid tumors, that remains to be shown in future studies, the investigators said. 

“Our results have important implications for the future of oncology care,” said Rocco, who is director of the Mass Eye and Ear/Mass General Head and Neck Molecular Oncology Research Laboratory and senior author of the Cancer paper. “MATH offers a simple, quantitative way to test hypotheses about intratumor genetic heterogeneity, including the likelihood that targeted therapy will succeed. The results also raise important questions about how genetic heterogeneity develops within a tumor and whether heterogeneity can be exploited therapeutically.”

The study was supported by National Institute of Dental and Craniofacial Research grants R01DE022087 and RC2DE020958, National Cancer Institute grant R21CA119591, Cancer Prevention Research Institute of Texas grant RP100233, and the Bacardi MEEI Biobank Fund.  Mass General has filed a patent application for the MATH measure.

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Mind-body Genomics

Mind-body Genomics

Relaxation response immediately alters gene expression tied to inflammation, metabolism and insulin

By SUE McGREEVEY
Wed, 2013-05-01

Image: National Center for Complementary and Alternative Medicine

A new study from investigators at the Benson-Henry Institute for Mind/Body Medicine at Massachusetts General Hospital and Beth Israel Deaconess Medical Center finds that eliciting the relaxation response—a physiologic state of deep rest induced by practices such as meditation, yoga, deep breathing and prayer—produces immediate changes in the expression of genes involved in immune function, energy metabolism and insulin secretion.

“Many studies have shown that mind/body interventions like the relaxation response can reduce stress and enhance wellness in healthy individuals and counteract the adverse clinical effects of stress in conditions like hypertension, anxiety, diabetes and aging,” said Herbert Benson, HMS professor of medicine at Mass General and co-senior author of the report.

Benson is director emeritus of the Benson-Henry Institute.

“Now for the first time we’ve identified the key physiological hubs through which these benefits might be induced,” he said.

Published in the open-access journal PLOS ONE, the study combined advanced expression profiling and systems biology analysis to both identify genes affected by relaxation response practice and to determine the potential biological relevance of those changes.

“Some of the biological pathways we identify as being regulated by relaxation response practice are already known to play specific roles in stress, inflammation and human disease. For others, the connections are still speculative, but this study is generating new hypotheses for further investigation," said Towia Libermann, HMS associate professor of medicine at Beth Israel Deaconess and co-senior author of the study.

Benson first described the relaxation response—the physiologic opposite of the fight-or-flight response—almost 40 years ago, and his team has pioneered the application of mind/body techniques to a wide range of health problems. Studies in many peer-reviewed journals have documented how the relaxation response both alleviates symptoms of anxiety and many other disorders and also affects factors such as heart rate, blood pressure, oxygen consumption and brain activity. 

In 2008, Benson and Libermann led a study finding that long-term practice of the relaxation response changed the expression of genes involved with the body’s response to stress. The current study examined changes produced during a single session of relaxation response practice, as well as those taking place over longer periods of time.

The study enrolled a group of 26 healthy adults with no experience in relaxation response practice, who then completed an 8-week relaxation-response training course.

Before they started their training, they went through what was essentially a control group session: Blood samples were taken before and immediately after the participants listened to a 20-minute health education CD and again 15 minutes later. After completing the training course, a similar set of blood tests was taken before and after participants listened to a 20-minute CD used to elicit the relaxation response as part of daily practice. 

The sets of blood tests taken before the training program were designated “novice,” and those taken after training completion were called “short-term practitioners.” For further comparison, a similar set of blood samples was taken from a group of 25 individuals with 4 to 25 years’ experience regularly eliciting the relaxation response through many different techniques before and after they listened to the same relaxation response CD.

Blood samples from all participants were analyzed to determine the expression of more than 22,000 genes at the different time points.

The results revealed significant changes in the expression of several important groups of genes between the novice samples and those from both the short- and long-term sets. Even more pronounced changes were shown in the long-term practitioners. 

A systems biology analysis of known interactions among the proteins produced by the affected genes revealed that pathways involved with energy metabolism, particularly the function of mitochondria, were upregulated during the relaxation response. Pathways controlled by activation of a protein called NF-κB—known to have a prominent role in inflammation, stress, trauma and cancer—were suppressed after relaxation response elicitation. The expression of genes involved in insulin pathways was also significantly altered.

“The combination of genomics and systems biology in this study provided great insight into the key molecules and physiological gene interaction networks that might be involved in relaying beneficial effects of relaxation response in healthy subjects,” said Manoj Bhasin, HMS assistant professor of medicine, co-lead author of the study, and co-director of the Beth Israel Deaconess Genomics, Proteomics, Bioinformatics and Systems Biology Center.

Bhasin noted that these insights should provide a framework for determining, on a genomic basis, whether the relaxation response will help alleviate symptoms of diseases triggered by stress. The work could also lead to developing biomarkers that may suggest how individual patients will respond to interventions.

Benson stressed that the long-term practitioners in this study elicited the relaxation response through many different techniques—various forms of meditation, yoga or prayer—but those differences were not reflected in the gene expression patterns.

“People have been engaging in these practices for thousands of years, and our finding of this unity of function on a basic-science, genomic level gives greater credibility to what some have called ‘new age medicine,’ ” he said.

“While this and our previous studies focused on healthy participants, we currently are studying how the genomic changes induced by mind/body interventions affect pathways involved in hypertension, inflammatory bowel disease and irritable bowel syndrome. We have also started a study—a collaborative undertaking between Dana-Farber Cancer Institute, Mass General and Beth Israel Deaconess—in patients with precursor forms of multiple myeloma, a condition known to involve activation of NF-κB pathways," said Libermann, who is the director of the Beth Israel Deaconess Medical Center Genomics, Proteomics, Bioinformatics and Systems Biology Center.

The study was supported by Centers for Disease Control and Prevention grants H75 CCH123424 and R01 DP000339, by National Center for Complementary and Alternative Medicine grant R01 AT006464-01, and by National Center for Research Resources grant M01 RR01032.

Adapted from a joint Mass General and Beth Israel Deaconess news release.

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