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Study Finds Genetic Mutations in Healthy Human Tissues

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General mutations throughout the body

The standard view of biology is that every normal cell copies its DNA instruction book with complete accuracy every time it divides. And thus, with a few exceptions like the immune system, cells in normal, healthy tissue continue to contain exactly the same genome sequence as was present in the initial single-cell embryo that gave rise to that individual. But new evidence suggests it may be time to revise that view.

By analyzing genetic information collected throughout the bodies of nearly 500 different individuals, researchers discovered that almost all had some seemingly healthy tissue that contained pockets of cells bearing particular genetic mutations. Some even harbored mutations in genes linked to cancer. The findings suggest that nearly all of us are walking around with genetic mutations within various parts of our bodies that, under certain circumstances, may have the potential to give rise to cancer or other health conditions.

Efforts such as NIH’s The Cancer Genome Atlas (TCGA) have extensively characterized the many molecular and genomic alterations underlying various types of cancer. But it has remained difficult to pinpoint the precise sequence of events that lead to cancer, and there are hints that so-called normal tissues, including blood and skin, might contain a surprising number of mutations —perhaps starting down a path that would eventually lead to trouble.

In the study published in Science, a team from the Broad Institute at MIT and Harvard, led by Gad Getz and postdoctoral fellow Keren Yizhak, along with colleagues from Massachusetts General Hospital, decided to take a closer look. They turned their attention to the NIH’s Genotype-Tissue Expression (GTEx) project.

The GTEx is a comprehensive public resource that shows how genes are expressed and controlled differently in various tissues throughout the body. To capture those important differences, GTEx researchers analyzed messenger RNA sequences within thousands of healthy tissue samples collected from people who died of causes other than cancer.

Getz, Yizhak, and colleagues wanted to use that extensive RNA data in another way: to detect mutations that had arisen in the DNA genomes of cells within those tissues. To do it, they devised a method for comparing those tissue-derived RNA samples to the matched normal DNA. They call the new method RNA-MuTect.

All told, the researchers analyzed RNA sequences from 29 tissues, including heart, stomach, pancreas, and fat, and matched DNA from 488 individuals in the GTEx database. Those analyses showed that the vast majority of people—a whopping 95 percent—had one or more tissues with pockets of cells carrying new genetic mutations.

While many of those genetic mutations are most likely harmless, some have known links to cancer. The data show that genetic mutations arise most often in the skin, esophagus, and lung tissues. This suggests that exposure to environmental elements—such as air pollution in the lung, carcinogenic dietary substances in the esophagus, or the ultraviolet radiation in sunlight that hits the skin—may play important roles in causing genetic mutations in different parts of the body.

The findings clearly show that, even within normal tissues, the DNA in the cells of our bodies isn’t perfectly identical. Rather, mutations constantly arise, and that makes our cells more of a mosaic of different mutational events. Sometimes those altered cells may have a subtle growth advantage, and thus continue dividing to form larger groups of cells with slightly changed genomic profiles. In other cases, those altered cells may remain in small numbers or perhaps even disappear.

It’s not yet clear to what extent such pockets of altered cells may put people at greater risk for developing cancer down the road. But the presence of these genetic mutations does have potentially important implications for early cancer detection. For instance, it may be difficult to distinguish mutations that are truly red flags for cancer from those that are harmless and part of a new idea of what’s “normal.”

To further explore such questions, it will be useful to study the evolution of normal mutations in healthy human tissues over time. It’s worth noting that so far, the researchers have only detected these mutations in large populations of cells. As the technology advances, it will be interesting to explore such questions at the higher resolution of single cells.

Getz’s team will continue to pursue such questions, in part via participation in the recently launched NIH Pre-Cancer Atlas. It is designed to explore and characterize pre-malignant human tumors comprehensively. While considerable progress has been made in studying cancer and other chronic diseases, it’s clear we still have much to learn about the origins and development of illness to build better tools for early detection and control.

Reference:

[1] RNA sequence analysis reveals macroscopic somatic clonal expansion across normal tissues. Yizhak K, Aguet F, Kim J, Hess JM, Kübler K, Grimsby J, Frazer R, Zhang H, Haradhvala NJ, Rosebrock D, Livitz D, Li X, Arich-Landkof E, Shoresh N, Stewart C, Segrè AV, Branton PA, Polak P, Ardlie KG, Getz G. Science. 2019 Jun 7;364(6444).

Links:

Genotype-Tissue Expression Program

The Cancer Genome Atlas (National Cancer Institute/NIH)

Pre-Cancer Atlas (National Cancer Institute/NIH)

Getz Lab (Broad Institute, Cambridge, MA)

NIH Support: Common Fund; National Heart, Lung, and Blood Institute; National Human Genome Research Institute; National Institute of Mental Health; National Cancer Institute; National Library of Medicine; National Institute on Drug Abuse; National Institute of Neurological Diseases and Stroke


Deciphering Another Secret of Life

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Srivatsan Raman
Credit: Robin Davies, University of Wisconsin-Madison

In 1953, Francis Crick famously told the surprised customers at the Eagle and Child pub in London that he and Jim Watson had discovered the secret of life. When NIH’s Marshall Nirenberg and his colleagues cracked the genetic code in 1961, it was called the solution to life’s greatest secret. Similarly, when the complete human genome sequence was revealed for the first time in 2003, commentators (including me) referred to this as the moment where the book of life for humans was revealed. But there are many more secrets of life that still need to be unlocked, including figuring out the biochemical rules of a protein shape-shifting phenomenon called allostery [1].

Among those taking on this ambitious challenge is a recipient of a 2018 NIH Director’s New Innovator Award, Srivatsan Raman of the University of Wisconsin-Madison. If successful, such efforts could revolutionize biology by helping us better understand how allosteric proteins reconfigure themselves in the right shapes at the right times to regulate cell signaling, metabolism, and many other important biological processes.

What exactly is an allosteric protein? Proteins have active, or orthosteric, sites that turn the proteins off or on when specific molecules bind to them. Some proteins also have less obvious regulatory, or allosteric, sites that indirectly affect the proteins’ activity when outside molecules bind to them. In many instances, allosteric binding triggers a change in the shape of the protein.

Allosteric proteins include oxygen-carrying hemoglobin and a variety of enzymes crucial to human health and development. In his work, Raman will start by studying a relatively simple bacterial protein, consisting of less than 200 amino acids, to understand the basics of how allostery works over time and space.

Raman, who is a synthetic biologist, got the idea for this project a few years ago while tinkering in the lab to modify an allosteric protein to bind new molecules. As part of the process, he and his team used a new technology called deep mutational scanning to study the functional consequences of removing individual amino acids from the protein [2].

The screen took them on a wild ride of unexpected functional changes, and a new research opportunity called out to him. He could combine this scanning technology with artificial intelligence and other cutting-edge imaging and computational tools to probe allosteric proteins more systematically in hopes of deciphering the basic molecular rules of allostery.

With the New Innovator Award, Raman’s group will first create a vast number of protein mutants to learn how best to determine the allosteric signaling pathway(s) within a protein. They want to dissect out the properties of each amino acid and determine which connect into a binding site and precisely how those linkages are formed. The researchers also want to know how the amino acids tend to configure into an inactive state and how that structure changes into an active state.

Based on these initial studies, the researchers will take the next step and use their dataset to predict where allosteric pathways are found in individual proteins. They will also try to figure out if allosteric signals are sent in one direction only or whether they can be bidirectional.

The experiments will be challenging, but Raman is confident that they will serve to build a more unified view of how allostery works. In fact, he hopes the data generated—and there will be a massive amount—will reveal novel sites to control or exploit allosteric signaling. Such information will not only expand fundamental biological understanding, but will accelerate efforts to discover new therapies for diseases, such as cancer, in which disruption of allosteric proteins plays a crucial role.

References:

[1] Allostery: an illustrated definition for the ‘second secret of life.’ Fenton AW. Trends Biochem Sci. 2008 Sep;33(9):420-425.

[2] Engineering an allosteric transcription factor to respond to new ligands. Taylor ND, Garruss AS, Moretti R, Chan S, Arbing MA, Cascio D, Rogers JK, Isaacs FJ, Kosuri S, Baker D, Fields S, Church GM, Raman S. Nat Methods. 2016 Feb;13(2):177-183.

Links:

Drug hunters explore allostery’s advantages. Jarvis LM, Chemical & Engineering News. 2019 March 10

Allostery: An Overview of Its History, Concepts, Methods, and Applications. Liu J, Nussinov R. PLoS Comput Biol. 2016 Jun 2;12(6):e1004966.

Srivatsan Raman (University of Wisconsin-Madison)

Raman Project Information (NIH RePORTER)

NIH Director’s New Innovator Award (Common Fund/NIH)

NIH Support: National Institute of General Medical Sciences; Common Fund


Personalized Combination Therapies Yield Better Cancer Outcomes

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Doctor consulting with patient
Credit: NIH National Cancer Institute Visuals Online/Daniel Sone

Gratifying progress has been made recently in an emerging area of cancer medicine called precision oncology. It’s a bold attempt to target treatment to the very genes and molecules driving a cancer, aiming to slow or even halt its growth. But there’s always more to learn. Now comes evidence that, while a single well-matched drug might be good, a tailored combination of drugs that attack a cancer in multiple ways at once might be even better.

The findings come from the I-PREDICT clinical trial, which treated people with advanced cancer who hadn’t benefited from previous therapy [1]. The NIH-funded team found that analyzing a tumor’s unique genetic and molecular profile provided enough information to recommend individualized combination therapies to patients. What’s more, patients who followed their individualized combination therapies most closely lived longer, with longer periods of progression-free disease, than did those who took fewer of the recommended drugs.

In most previous clinical trials of precision oncology, researchers have relied on a tumor’s unique profile to identify a single, well-matched drug to treat each patient. But cancer is complex, and, just as with certain infectious diseases, tumors commonly develop resistance to a single drug.

In the trial reported in Nature Medicine, researchers led by Razelle Kurzrock and Jason Sicklick, University of California, San Diego, wondered if they could improve treatment responses by tailoring combinations of cancer drugs to target as many molecular and genetic changes in a person’s cancer as possible.

To test the potential for this strategy to work, the researchers enrolled 83 people with various cancers that had advanced despite previous treatment. Tumor tissue from each patient was run through a comprehensive battery of tests, and researchers sequenced hundreds of genes to look for telltale alterations in their DNA.

They also looked for evidence that a cancer had defects affecting the DNA “mismatch repair” pathway, which causes some tumors to generate larger numbers of mutations than others. Mismatch repair defects have been shown to predict better responses to immunotherapies, which are designed to harness the immune system against cancer .

With all the data in hand, a special panel of oncologists, pharmacologists, cancer biologists, geneticists, surgeons, radiologists, pathologists, and bioinformatics experts consulted to arrive at the right customized combination of drugs for each patient.

The panel’s findings were presented to the health care team working with each patient. The physician for each patient then had the final decision on whether to recommend the treatment regimen, balancing the panel’s suggestions with other real-world factors, such as a patient’s insurance coverage, availability of drugs, and his or her treatment preference.

Ten patients decided to stick with unmatched treatment. But 73 participants received a customized combination therapy. As no two molecular profiles were identical, the customized treatment regimens varied from person to person.

Many people received designer drugs targeting particular genetic alterations. Some also received checkpoint inhibitor immunotherapies to unleash the immune system against cancer. Four people also were treated with hormone therapies in combination with molecularly targeted drugs. In all, most regimens combined two to five drugs to target each cancer profile.

Participants were followed until their cancer progressed, they could no longer take treatment, or they died. For each person, the researchers calculated a “matching score,” roughly defined as the number of molecular alterations matched to administered drug(s), with some further calculations.

The evidence showed that those with matching scores greater than 50 percent, meaning more than half of a tumor’s identified aberrations had been targeted, were more likely to have stopped the progression of their cancers. Importantly, half of patients with the higher matching scores had prolonged stable disease (six months or longer) or a complete or partial remission. Similar results were attained in only 22 percent of those with low or no matching scores.

These encouraging results suggest that customized combinations of targeted treatments will help to advance precision oncology. However, there are still many challenges. For example, many of the combinations used in the study have not yet been safety tested. The researchers managed the potential risk of toxicities by starting patients on an initial low dose and having their physicians follow them closely while the dose was increased to a level well-tolerated by each individual patient.

And indeed, they saw no evidence that those receiving a greater proportion of “matched” drugs (i.e. those with a higher matching score) were more likely to experience adverse effects than those who took fewer drugs. So, that’s an encouraging sign.

The researchers are now enrolling patients in a new version of the I-PREDICT trial. Unlike the initial plan, patients are now being enrolled prior to receiving any treatment for a recently diagnosed aggressive, often-lethal form of cancer. The hope is that treating patients with well-matched, multi-drug treatment combinations early will yield even better results than waiting until standard treatment has failed. If correct, it would mark significant progress in building the future of precision oncology.

Reference:

[1] Molecular profiling of cancer patients enables personalized combination therapy: the I-PREDICT study. Sicklick JK, Kato S, Okamura R, Schwaederle M, Hahn ME, Williams CB, De P, Krie A, Piccioni DE, Miller VA, Ross JS, Benson A, Webster J, Stephens PJ, Lee JJ, Fanta PT, Lippman SM, Leyland-Jones B, Kurzrock R. Nat Med. 2019 Apr 22.

Links:

Precision Medicine in Cancer Treatment (National Cancer Institute/NIH)

Study of Molecular Profile-Related Evidence to Determine Individualized Therapy for Advanced or Poor Prognosis Cancers (I-PREDICT) (Clinicaltrials.gov)

Razelle Kurzrock (University of California, San Diego)

Jason Sicklick (University of California, San Diego)

NIH Support: National Cancer Institute


Study Finds No Benefit for Dietary Supplements

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Eating healthy
Credit: iStock/Artfully79

More than half of U.S. adults take dietary supplements [1]. I don’t, but some of my family members do. But does popping all of these vitamins, minerals, and other substances really lead to a longer, healthier life? A new nationwide study suggests it doesn’t.

Based on an analysis of survey data gathered from more than 27,000 people over a six-year period, the NIH-funded study found that individuals who reported taking dietary supplements had about the same risk of dying as those who got their nutrients through food. What’s more, the mortality benefits associated with adequate intake of vitamin A, vitamin K, magnesium, zinc, and copper were limited to food consumption.

The study, published in the Annals of Internal Medicine, also uncovered some evidence suggesting that certain supplements might even be harmful to health when taken in excess [2]. For instance, people who took more than 1,000 milligrams of supplemental calcium per day were more likely to die of cancer than those who didn’t.

The researchers, led by Fang Fang Zhang, Tufts University, Boston, were intrigued that so many people take dietary supplements, despite questions about their health benefits. While the overall evidence had suggested no benefits or harms, results of a limited number of studies had suggested that high doses of certain supplements could be harmful in some cases.

To take a broader look, Zhang’s team took advantage of survey data from tens of thousands of U.S. adults, age 20 or older, who had participated in six annual cycles of the National Health and Nutrition Examination Survey (NHANES) between 1999-2000 and 2009-2010. NHANES participants were asked whether they’d used any dietary supplements in the previous 30 days. Those who answered yes were then asked to provide further details on the specific product(s) and how long and often they’d taken them.

Just over half of participants reported use of dietary supplements in the previous 30 days. Nearly 40 percent reported use of multivitamins containing three or more vitamins.

Nutrient intake from foods was also assessed. Each year, the study’s participants were asked to recall what they’d eaten over the last 24 hours. The researchers then used that information to calculate participants’ nutrient intake from food. Those calculations indicated that more than half of the study’s participants had inadequate intake of vitamins D, E, and K, as well as choline and potassium.

Over the course of the study, more than 3,600 of the study’s participants died. Those deaths included 945 attributed to cardiovascular disease and 805 attributed to cancer. The next step was to look for any association between the nutrient intake and the mortality data.

The researchers found the use of dietary supplements had no influence on mortality. People with adequate intake of vitamin A, vitamin K, magnesium, zinc, and copper were less likely to die. However, that relationship only held for nutrient intake from food consumption.

People who reported taking more than 1,000 milligrams of calcium per day were more likely to die of cancer. There was also evidence that people who took supplemental vitamin D at a dose exceeding 10 micrograms (400 IU) per day without a vitamin D deficiency were more likely to die from cancer.

It’s worth noting that the researchers did initially see an association between the use of dietary supplements and a lower risk of death due to all causes. However, those associations vanished when they accounted for other potentially confounding factors.

For example, study participants who reported taking dietary supplements generally had a higher level of education and income. They also tended to enjoy a healthier lifestyle. They ate more nutritious food, were less likely to smoke or drink alcohol, and exercised more. So, it appears that people who take dietary supplements are likely to live a longer and healthier life for reasons that are unrelated to their supplement use.

While the study has some limitations, including the difficulty in distinguishing association from causation, and a reliance on self-reported data, its findings suggest that the regular use of dietary supplements should not be recommended for the general U.S. population. Of course, this doesn’t rule out the possibility that certain subgroups of people, including perhaps those following certain special diets or with known nutritional deficiencies, may benefit.

These findings serve up a reminder that dietary supplements are no substitute for other evidence-based approaches to health maintenance and eating nutritious food. Right now, the best way to live a long and healthy life is to follow the good advice offered by the rigorous and highly objective reviews provided by the U.S. Preventive Services Task Force [3]. Those tend to align with what I hope your parents offered: eat a balanced diet, including plenty of fruits, veggies, and healthy sources of calcium and protein. Don’t smoke. Use alcohol in moderation. Avoid recreational drugs. Get plenty of exercise.

References:

[1] Trends in Dietary Supplement Use Among US Adults From 1999-2012. Kantor ED, Rehm CD, Du M, White E, Giovannucci EL. JAMA. 2016 Oct 11;316(14):1464-1474.

[2] Association among dietary supplement use, nutrient intake, and mortality among U.S. adults. Chen F, Du M, Blumberg JB, Ho Chui KK, Ruan M, Rogers G, Shan Z, Zeng L, Zhang. Ann Intern Med. 2019 Apr 9. [Epub ahead of print].

[3] Vitamin Supplementation to Prevent Cancer and CVD: Preventive Medication. U.S. Preventive Services Task Force, February 2014.

Links:

Office of Dietary Supplements (NIH)

Healthy Eating Plan (National Heart, Lung, and Blood Institute/NIH)

National Health and Nutrition Examination Survey (Centers for Disease Control and Prevention, Atlanta)

U.S. Preventive Services Task Force (Rockville, MD)

Fang Fang Zhang (Tufts University, Boston)

NIH Support: National Institute on Minority Health and Health Disparities


New Target for Cancer Immunotherapy: Exosomes

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It was once a central tenet of biology that RNA molecules did their work inside the cell. But it’s now clear that RNA molecules are also active outside the cell, with potentially major implications for our health. To learn more about these unrecognized roles, the NIH Common Fund has launched the Extracellular RNA (exRNA) Communication Program.

This month, members of this research consortium described their latest progress in unraveling the secrets of exRNA in a group of 18 papers in the Cell family of journals. And it’s not just RNA that the consortium is studying, it’s also proteins. Among the many exciting results just published is the serendipitous discovery that proteins carried inside tiny, bubble-like vesicles, called exosomes, may influence a cancer’s response to immunotherapy [1]. The work sheds light on why certain cancers are resistant to immunotherapy and points to new strategies for unleashing the immune system in the fight against cancer.

The new findings center on a type of immunotherapy drugs known as checkpoint inhibitors. They are monoclonal antibodies produced by industry that can boost the immune system’s ability to attack and treat cancer.

One of those antibodies specifically targets a protein, called PD-1, on the surface of certain immune cells. When PD-1 binds a similarly named protein, called PD-L1, on the surface of another cell, the interaction prevents immune cells from attacking. Some tumors seem to have learned this and load up on PD-L1 to evade the immune system.

That’s where checkpoint inhibitors come in. By blocking the interaction between PD-1 and PD-L1, the treatment removes a key check on the immune system, allowing certain immune cells to wake up and attack the tumor.

Checkpoint inhibitors work better in some cancer types than in others. In melanoma, for example, up to about 30 percent of patients respond to checkpoint inhibitor therapy. But in prostate cancer, response rates are in the single digits.

Researchers led by Robert Blelloch, a member of the exRNA consortium and a scientist at the University of California, San Francisco, wanted to know why. He and his team looked for clues in RNA within the cells taken from immunotherapy-resistant prostate cancers.

As published in Cell, the researchers got their first hint of something biologically intriguing in an apparent discrepancy in their data. As they expected from prior work, PD-L1 protein was present in the treatment-resistant cancers. But the PD-L1 messenger RNAs (mRNA), which serve as templates for producing the protein, told an unexpected story. The resistant cancer cells made far more PD-L1 mRNAs than needed to produce the modest levels of PD-L1 proteins detected inside the cells.

Where was the missing PD-L1? Blelloch’s team found it in exosomes. The cancer cells were packaging large quantities of the protein inside exosomes and secreting them out of the cell to other parts of the body.

In additional studies with a mouse model of prostate cancer, the researchers found that those PD-L1-packed exosomes travel through the blood and lymphatic systems to lymph nodes, the sites where immune cells become activated. Once there, PD-L1-laden exosomes put the immune system to sleep, preventing certain key cells from locating and attacking the cancer, including the primary tumor and places where it may have spread.

In important follow up studies, the researchers edited two genes in cancer cells to prevent them from producing exosomes. And, in the absence of exosomes, the cells no longer formed tumors. Importantly, both edited and unedited cells still produced PD-L1, but only those that exported PD-L1 in exosomes disarmed the immune system. Studies in a mouse model of immunotherapy-resistant colorectal cancer yielded similar results.

The new evidence suggests that blocking the release of PD-L1 in exosomes, even temporarily, might allow the immune system to launch a successful and sustained attack against a cancer.

Blelloch notes that many intriguing questions remain. For example, it’s not yet clear why antibodies that target PD-L1 on cancer cells don’t disable PD-L1 found in exosomes. The good news is that the new findings suggest it may be possible to find small molecules that do target PD-L1-packed exosomes, unleashing the immune system against cancers that don’t respond to existing checkpoint inhibitors. In fact, Blelloch’s team is already screening for small molecules that might fit the bill.

Since its launch about five years ago, the exRNA Communication Program has published an impressive 480 peer-reviewed papers, including the latest work in the Cell family of journals. I’d encourage readers to click on some of the other excellent work. I hear that another batch of papers will be published later this year.

Reference:

[1] Suppression of exosomal PD-L induces systemic anti-tumor immunity and memory. Poggio M, Hu T, Pai CC, Chu B, Belair CD, Chang A, Montabana E, Lang UE, Fu Q, Fong L, Blelloch R. Cell. 2019 Apr 4;177(2):414-427.

Links:

Video: Unlocking the Mysteries of RNA Communication (Common Fund/NIH)

Immunotherapy to Treat Cancer (National Cancer Institute/NIH)

Blelloch Lab (University of California, San Francisco)

NIH Support: Common Fund; National Cancer Institute; National Center for Advancing Translational Sciences; National Heart, Lung, and Blood Institute; National Institute on Drug Abuse


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