Melanoma – the deadliest and most aggressive form of skin cancer – has long been linked to time spent in the sun. Now a team led by scientists from the Broad Institute and Dana-Farber Cancer Institute has sequenced the whole genomes of 25 metastatic melanoma tumors, confirming the role of chronic sun exposure and revealing new genetic changes important in tumor formation.

In an article published online May 9 in Nature, the authors provide the first high-resolution view of the genomic landscape of human melanoma tumors. Previous genetic analyses have focused on the exomes of many types of cancer tumors, concentrating on the tiny fraction of the genome that provides the genetic code for producing proteins. Whole genomes contain a wealth of genetic information, and by sequencing and analyzing 25 metastatic melanoma tumors – a significant technical and computational feat – scientists can learn vastly more about the variety of genetic alterations that matter in melanoma.

“Sequencing the whole genome certainly adds a richness of discovery that can’t be fully captured with a whole exome,” said Levi A. Garraway, a senior associate member of the Broad Institute, an associate professor at Dana-Farber Cancer Institute and Harvard Medical School, and co-senior author of the paper.

“By looking across the entire genome you can more accurately determine the background mutation rate and the different classes of mutations, and more confidently describe the pattern of ultraviolet-induced mutagenesis in melanoma,” said Michael F. Berger, co-first author of the paper. He worked in the Broad’s cancer genome analysis group and with Garraway as a research scientist and computational biologist before moving to Memorial Sloan-Kettering Cancer Center.

When the scientists explored the whole genome data generated and analyzed at the Broad, they found that the rates of genetic mutations rose along with chronic sun exposure in patients, confirming the role of sun damage in disease development.

“Whole-genome analysis of human melanoma tumors shows for the first time the existence of many structural rearrangements in this tumor type,” said Lynda Chin, a senior associate member of the Broad and co-senior author of the paper. Formerly at Dana-Farber and Harvard Medical School, she is now chair of the Department of Genomic Medicine at the University of Texas MD Anderson Cancer Center.

As expected, the scientists detected known BRAF and NRAS mutations in 24 of the 25 tumors. Both genes are involved in sending signals important in cell growth.

One other gene leaped out: PREX2, previously implicated in breast cancer for blocking a tumor-suppressor pathway, was altered in 44 percent of patients. In a larger validation cohort of 107 tumors, the frequency of the mutation was 14 percent.

PREX2 is mutated in a convergence of genetic disruption that appears to accelerate tumor development. Its mutations occurred not just at hot spots that typically turn on an oncogene, a type of cancer-causing gene, and drive cancer forward. The alterations were also scattered across the length of the gene in a pattern typically seen when another type of cancer-causing gene, known as tumor suppressors, are turned off.

“The pattern of mutations here looks a lot more like a tumor suppressor gene, but from the functional experiments, it behaved more like an oncogene,” Berger said.

When PREX2 functions normally, it interacts with the protein PTEN. PTEN is well known as a tumor suppressor, controlling growth in normal cells. Mouse experiments in Chin’s lab at Dana-Farber showed that PREX2 mutations spurred tumor growth in ways that are not fully understood.

“We still can’t say we know exactly how it works,” Garraway said. “PREX2 may be in a very interesting new category of mutated cancer genes that point us to at least one and maybe more pathways that would be worth targeting therapeutically in melanoma.”

The identification of PREX2 may be the tip of the iceberg.

“New melanoma genes remain to be discovered by this unbiased approach, as illustrated by the discovery of PREX2 and the demonstration of its oncogenicity in vivo,” said Chin.

More information: Berger, Hodis, Heffernan, Deribe, Lawrence et al. “Melanoma genome sequencing reveals frequent PREX2 mutations.” Nature, May 10, 2012. doi:10.1038/nature11071

Provided by Massachusetts Institute of Technology (news : web)

STANFORD, Calif. — The cells that slough off from a cancerous tumor into the bloodstream are a genetically diverse bunch, Stanford University School of Medicine researchers have found. Some have genes turned on that give them the potential to lodge themselves in new places, helping a cancer spread between organs. Others have completely different patterns of gene expression and might be more benign, or less likely to survive in a new tissue. Some cells may even express genes that could predict their response to a specific therapy. Even within one patient, the tumor cells that make it into circulating blood vary drastically.

The finding underscores how multiple types of treatment may be required to cure what appears outwardly as a single type of cancer, the researchers say. And it hints that the current cell-line models of human cancers, which showed patterns that differed from the tumor cells shed from human patients, need to be improved upon.

The new study, which will be published online May 7 in PLoS ONE, is the first to look at so-called circulating tumor cells one by one, rather than taking the average of many of the cells. And it’s the first to show the extent of the genetic differences between such cells.

“Within a single blood draw from a single patient, we’re seeing heterogeneous populations of circulating tumor cells,” said senior study author Stefanie Jeffrey, MD, professor of surgery and chief of surgical oncology research.

For over a century, scientists have known that circulating tumor cells, or CTCs, are shed from tumors and move through the bloodstreams of cancer patients. And over the past five years, there’s been a growing sense among many cancer researchers that these cells — accessible by a quick blood draw — could be the key to tracking tumors non-invasively. But separating CTCs from blood cells is hard; there can be as few as one or two CTCs in every milliliter of a person’s blood, mixed among billions of other blood cells.

To make their latest discovery, Jeffrey, along with an interdisciplinary team of engineers, quantitative biologists, genome scientists and clinicians, relied on a technology they developed in 2008. Called the MagSweeper, it’s a device that lets them isolate live CTCs with very high purity from patient blood samples, based on the presence of a particular protein — EpCAM — that’s on the surface of cancer cells but not healthy blood cells.

With the goal of studying CTCs from breast cancer patients, the team first tested whether they could accurately detect the expression levels of 95 different genes in single cells from seven different cell-line models of breast cancer — a proof of principle since they already knew the genetics of these tumors. These included four cell lines generally used by breast cancer researchers and pharmaceutical scientists worldwide and three cell lines specially generated from patients’ primary tumors.

“Most researchers look at just a few genes or proteins at a time in CTCs, usually by adding fluorescent antibodies to their samples consisting of many cells,” said Jeffrey. “We wanted to measure the expression of 95 genes at once and didn’t want to pool our cells together, so that we could detect differences between individual tumor cells.”

So once Jeffrey and her collaborators isolated CTCs using the MagSweeper, they turned to a different kind of technology: real-time PCR microfluidic chips, invented by a Stanford collaborator, Stephen Quake, PhD, professor of bioengineering. They purified genetic material from each CTC and used the high-throughput technology to measure the levels of all 95 genes at once. The results on the cell-line-derived cells were a success; the genes in the CTCs reflected the known properties of the mouse cell-line models. So the team moved on to testing the 95 genes in CTCs from 50 human breast cancer patients — 30 with cancer that had spread to other organs, 20 with only primary breast tumors.

“In the patients, we ended up with 32 of the genes that were most dominantly expressed,” said Jeffrey. “And by looking at levels of those genes, we could see at least two distinct groups of circulating tumors cells.” Depending on which genes they used to divide the CTCs into groups, there were as many as five groups, she said, each with different combinations of genes turned on and off. And if they’d chosen genes other than the 95 they’d picked, they likely would have seen different patterns of grouping. However, because the same individual CTCs tended to group together in multiple different analyses, these cells likely represent different types of spreading cancer cells.

The diversity, Jeffrey said, means that tumors may contain multiple types of cancer cells that may get into the bloodstream, and a single biopsy from a patient’s tumor doesn’t necessarily reflect all the molecular changes that are driving a cancer forward and helping it spread. Moreover, different cells may require different therapies. One breast cancer patient studied, for example, had some CTCs positive for the marker HER2 and others lacked the marker. When the patient was treated with a drug designed to target HER2-positive cancers, the CTCs lacking the molecule remained in her bloodstream.

When the team went on to compare the diverse genetic profiles of the breast cancer patients’ CTCs with the cells they’d studied from the cell lines, they were in for another surprise: None of the human CTCs had the same gene patterns as any of the cell-line models.

“These models are what people are using for drug discovery and initial drug testing,” said Jeffrey, “but our finding suggests that perhaps they’re not that helpful as models of spreading cancers.” While the human cell-line cells did show diversity between each of the seven cell lines, they didn’t fall into any of the same genetic profiles as the CTCs from human blood samples.

These results don’t have immediate impacts for cancer patients in the clinic because more work is needed to discover whether different types of CTCs respond to different therapies and whether that will be clinically useful for guiding treatment decisions. But the finding is a step forward in understanding the basic science behind the bits of tumors that circulate in the blood. It’s the first time that scientists have used high-throughput gene analysis to study individual CTCs, and opens the door for future experiments that delve even more into the cell diversity. The Stanford team is now working on different methods of using CTCs for drug testing as well as studying the relationship between CTC genetic profiles and cancer treatment outcomes. They’ve also expanded their work to include primary lung and pancreatic cancers as well as breast tumors.

“We’re finding clinically relevant information in the tumor samples we’re sequencing for discovery-oriented research studies,” says Elaine Mardis, PhD, co-director of The Genome Institute at the School of Medicine. “Genome analysis can play a role at multiple time points during a patient’s treatment, to identify ‘driver’ mutations in the tumor genome and to determine whether cells carrying those mutations have been eliminated by treatment.”

This work is helping to guide the design of future cancer clinical trials in which treatment decisions are based on results of sequencing, says Mardis, who is speaking April 1 at the opening plenary session of the American Association for Cancer Research annual meeting in Chicago. She also is affiliated with the Siteman Cancer Center at the School of Medicine and Barnes-Jewish Hospital.

To date, Mardis and her colleagues have sequenced all the DNA — the genome — of tumor cells from more than 700 cancer patients. By comparing the genetic sequences in the tumor cells to healthy cells from the same patient, they can identify mutations underlying each patient’s cancer.

Already, information gleaned through whole-genome sequencing is pushing researchers to reclassify tumors based on their genetic makeup rather than their location in the body. In patients with breast cancer, for example, Mardis and her colleagues have found numerous driver mutations in genes that have not previously been associated with breast tumors.

A number of these genes have been identified in prostate, colorectal, lung or skin cancer, as well as leukemia and other cancers. Drugs that target mutations in these genes, including imatinib, ruxolitinib and sunitinib, while not approved for breast cancer, are already on the market for other cancers.

“We are finding genetic mutations in multiple tumor types that could potentially be targeted with drugs that are already available,” Mardis says.

She predicts, however, that it may require a paradigm change for oncologists to evaluate the potential benefits of individualized cancer therapy. While clinical trials typically involve randomly assigning patients to a particular treatment regimen, a personalized medicine approach calls for choosing drugs based on the underlying mutations in each patient’s tumor.

“Having all treatment options available for every patient doesn’t fit neatly into the confines of a carefully designed clinical trial,” Mardis acknowledges. “We’re going to need more flexibility.”

When during the course of cancer mutations develop also is likely to be important in decisions about treatment. In a recent study, Mardis and her team mapped the genetic evolution of leukemia and found clues to suggest that targeted cancer drugs should be aimed at mutations that develop early in the course of the disease.

Using “deep digital sequencing,” a technique developed at The Genome Institute, they sequenced individual mutations in patients’ tumor samples more than 1,000 times each. This provides a read-out of the frequency of each mutation in a patient’s tumor genome and allowed the researchers to map the genetic evolution of cancer cells as the disease progressed.

They found that as cancer evolves, tumors acquire new mutations but always retain the original cluster of mutations that made the cells cancerous in the first place. Their discovery suggests that drugs targeted to cancer may be more effective if they are directed toward genetic changes that occur early in the course of cancer. Drugs that target mutations found exclusively in later-evolving cancer cells likely may not have much effect on the disease because they would not kill all the tumor cells.

Mardis says that sequencing the entire genome of cancer cells is essential to piecing together an accurate picture of the way cancer cells evolve. If the researchers had sequenced only the small portion of the genome that involves genes, they would not have had the statistical power to track the frequency of mutations over time. (Only 1 to 2 percent of the genome consists of genes.)

In another study, a phase III clinical trial of post-menopausal women with estrogen-receptor positive breast cancer, the Washington University researchers have shown that sequencing can help to predict which women will respond to treatment with aromatase inhibitors. These estrogen-lowering drugs are often prescribed to shrink breast tumors before surgery. But only about half of women with estrogen-receptor positive breast cancer respond to these drugs, and doctors have not been able to predict which patients will benefit.

Interestingly, by sequencing patients’ breast tumors before and after aromatase inhibitor therapy, the researchers identified substantive genomic changes that had occurred in responsive patients, whereas the genomes of unresponsive patients remained largely unchanged by the therapy.

“No one has ever looked at treatment response at this level of resolution,” Mardis says. “It’s so obvious who is responding.”

In addition, the researchers have identified a series of mutations in the breast tumors that have corresponding small-molecule inhibitor drugs that target defective proteins. This finding indicates that for women who are not responding to aromatase inhibitors, treatment options may include combining conventional chemotherapy with the indicated small-molecule inhibitor.

“We felt it was important to show there could be therapeutic options available to patients who are resistant to aromatase inhibitors,” Mardis says. “As we move forward, we think sequencing will contribute crucial information to determining the best treatment options for patients.”

The research is funded by the National Cancer Institute, the National Human Genome Research Institute and the National Heart, Lung and Blood Institute, all of the National Institutes of Health, and the Washington University Cancer Genome Initiative.

A study by researchers at Indiana University, Methodist Research Institute that is published in The International Journal of Oncology reveals that a non-toxic, botanical orally administered formula controls aggressive human prostate tumors in mice.

The peer-reviewed pre-clinical in vivo study demonstrated that the prostate formula substantially suppresses tumor growth in aggressive, hormone-refractory (androgen-independent) human prostate cancer cells without any toxic side effects, even when administered at high doses. The formula is a combination of botanical extracts, phytonutrients, botanically enhanced medicinal mushrooms and antioxidants.

Inventor of the formula, Dr. Isaac Eliaz declared:

“This study is a milestone in the research of this formula, demonstrating its safety and effectiveness in treating human prostate cancer in an animal model. These positive results offer a significant contribution to prostate cancer research and add to the growing body of published data substantiating the role of natural compounds in the treatment of prostate cancer.”

This is the third study from a major university which has demonstrated the formula’s ability to suppress tumor growth and metastasis.

Leading researcher Dr. Daniel Silva remarks:

“Multiple studies have demonstrated that this prostate formula is a possible treatment for hormone-refractory prostate cancer.”

The study results showed that in comparison to controls, the formula managed to suppress tumor growth by 27% and substantially reduced the size of the tumor. It also blocked several genes, i.e. IGF2, NRNF2 and PLAU/uPA, which encourage cancer proliferation and metastasis. It furthermore increased CDKN1A expression, a gene that fights prostate cancer by blocking cellular mechanisms that promote cancer.

All of these characteristics prove that the formula has multiple anti-cancer mechanisms and genetic targets and confirm earlier published in vitro data that showed that the formula lowers the expression of PLAU/uPA genes in aggressive, hormone-independent prostate cancer cells.

The formula was previously studied at Columbia University and the Cancer Research Laboratory, Methodist Research Institute, at Indiana University Health, where the findings were also confirmed.

Written by Petra Rattue

A study led by Dr Janet Shipley from The Institute of Cancer Research (ICR) in London in collaboration with Dr Mauro Delorenzi from the SIB Swiss Institute of Bioinformatics in Lausanne has shown that a simple genetic test could help predict the aggressiveness of rhabdomyosarcoma tumours in children. The test, which should be introduced into clinical practice, would lead to changes in treatment for many patients, allowing some children to escape potentially long-term side-effects whilst giving others the intense treatments they need to increase their chances of survival. The results of the study are published online today in the Journal of Clinical Oncology.

Until now, the PAX3/FOXO1 fusion gene only served as a classification agent for tumour histology but never as a prognostic indicator. The research team found that children who have a tumour called rhabdomyosarcoma with this particular genetic fault have significantly poorer survival rates than other rhabdomyosarcoma patients. This fusion gene can thus be very useful in the prognosis of patient’s survival.

More than that, it can provide better information about how aggressively the tumour is likely to behave and help doctors to tailor treatment for each patient. So far, children diagnosed with rhabdomyosarcoma were treated with a combination of chemotherapy and surgery and sometimes radiotherapy. These treatments have helped improve survival rates, but they can also cause serious and long-term side-effects including the potential to develop another cancer later in life. But not all patients need such intense treatment. Dr Shipley says: “Our previous studies have raised issues with the current system of predicting patients’ risk, which is based on the appearance of patients’ tumours. Our new study finds that a simple genetic test should be incorporated into standard clinical practice as it significantly improves our ability to predict tumour aggressiveness. This fusion gene test could be used alongside other standard clinical measures to divide patients into one of four risk-groups, so that treatment can be tailored accordingly. Importantly, this will mean some patients who were previously categorised as high-risk could be able to avoid the side-effects associated with intense treatment, while others should receive the intense treatment they need to increase their chance of survival.”

The study required high level statistics expertise

To analyse the data for thousands of genes from 225 rhabdomyosarcoma samples, Dr Shipley called onto the expertise of the Bioinformatics Core Facility Group at the SIB Swiss Institute of Bioinformatics in Lausanne, which is led by Dr. Mauro Delorenzi. This group provides statistical and analysis support for either national and international academic and private teams. Dr. Edoardo Missiaglia and Dr. Pratyaksha Wirapati performed the analysis of the data provided in the frame of this study and constructed and evaluated systems to score the aggressiveness of the individual case of rhabdomyosarcoma. Their work allowed to identify a panel of 15 genes whose altered activity level could be used to predict how patients responded to treatment. However, it was also found that most of these gene changes are linked to the presence of the PAX3/FOXO1 fusion gene: the detection of which is much simpler and cheaper than that of altered gene activity levels. Dr Delorenzi says: “We showed that by making a good use of the information about the presence or absence of the fusion of the two gene PAX3 and FOXO1, alongside other standard clinical measures, we could create a risk scoring system that is very informative on the aggressiveness of a tumour; it is so good that the additional use of the complex gene activity information does not appear to help to further improve it.”

Using the new system, 31 per cent of patients in the study who would previously have been classified as intermediate risk would be reassigned to a lower risk group, while a further 29 per cent of intermediate-risk patients would be moved to a higher risk group. Combining the fusion gene test with two existing standard measures of risk for rhabdomyosarcomas – the patient’s age at diagnosis and the tumour’s stage of development – gave a simple but highly effective prognostic test.

The research team now intends to validate their findings using a larger European and independent data set. If confirmed, their method could be used in future clinical trials to assist clinicians in treatment decision. Dr Missiaglia adds: “In the same work we also show evidence that the gene activity information of 5 other genes might give important additional information in a subgroup, but since this is rare we do not yet have enough cases to be sure and this should be further tested on new data that are not yet available”.

Provided by Swiss Institute of Bioinformatics

Technology has the potential to transform our concept of sickness. An expert explains what the future holds

By Lucy McKeon

Lucy McKeon is an editorial fellow at Salon. More Lucy McKeon

• By Thomas Goetz
• Email Author |
• January 31, 2012 |
• 12:30 pm |
• Wired February 2012

We understand digitizing a book, but what does it mean to digitize a human being? asks Eric Topol.
Photo: Gregg Segal

Medicine has certainly progressed in the past 50 years, but the day when tricorders diagnose every ailment instantly and treatments are tailored to our DNA seems as far off as ever. Eric Topol is trying to bridge that gap. In his new book, The Creative Destruction of Medicine, Topol—the chief academic officer at Scripps Health—calls on patients to demand true digital medicine now. We talked to him about genetics, gadgets, and his vision of a Khan Academy for doctors.

Wired: Not many doctors get to take the stage at the Consumer Electronics Show, as you did in 2010. What was that like?

Eric Topol: It was a revelation. Normally people go to CES to learn about gizmos like HDTVs. And here I come to do a demo of wireless devices for health. The reaction was astounding: They began clapping when this little device I was holding showed an ultrasound of my heart on the big screen. It made me realize that consumers want to care about their health. They just need to get activated.

Wired: And that starts with this nifty concept you have of digitizing medicine.

Topol: Right. We understand digitizing a book, but what does it mean to digitize a human being? When I went to medical school, the term digital applied only to rectal exams. But today you can get a DNA sequence, you can get biosensors that record nearly every physiologic metric from blood pressure to brain waves, you can get a digital scan of any part of the body. These tools offer a window into each person that was unfathomable a few years ago.

Wired: But it’s not just the body; this scales up to the entire infrastructure of medicine.

Topol: That’s right. The digital world—the Internet and the cloud and supercomputing and social networking—is breaking medicine out of its cocoon. It’s a superconvergence we’ve seen in other walks of life but not in the health and medical sphere.

Wired: So what does digitized medicine get us?

Topol: We can start capturing people’s health data throughout their lives—all the little things that have lasting implications. For instance, we can track cumulative radiation exposure from every scan and x-ray. And consider the risk of drug interactions: Every year hundreds of thousands of Americans wind up in hospitals or worse because we didn’t match up the patient genomically with the right drug or dosage. Just capturing those things could save thousands of lives.

Wired: How do we make this happen now, rather than just waiting for a new, net-savvy generation of doctors?

Topol: We need a Khan Academy for doctors: captivating 15-minute videos on genomics, on wireless sensors, on advanced imaging, on health information systems. These things can revive the excitement they felt as premeds, when they first decided to go into this field. If we can get practicing physicians up to speed and really inspired, maybe we won’t have to wait a generation. I shudder to think about waiting 10 or 20 years for this transformation to occur.

Wired: But there are obstacles. For instance, many people in the tech world are afraid of running into bottlenecks getting FDA approval for new medical devices.

Topol: The FDA is moving very slowly, with considerable restraint and resistance. That’s one reason the technology is years behind where it should be. One example is a device called AliveCor. It’s a couple of sensors on a case that you can put on the back of an iPhone or a Droid phone to get your electrocardiogram and heart rhythm. It’s very inexpensive—less than $100. You can even send the results to your Facebook friends. In Europe it’s already approved and available today. But not in the US. A lot of these great, innovative ideas like sensors or rapid point-of-care geno-typing are moving slowly through the process with a considerable lack of support, as I see it. And these are largely just diagnostic tools, not therapeutics.

Wired: Meaning that they’re not doing anything to your body; they’re just taking information.

Topol: Exactly. A perfect example would be the glucose sensor that you can put on and get a reading every five minutes.

Wired: Which has likewise been hung up in FDA limbo.

Topol: Yeah. If you’re a diabetic and you’re using a glucose sensor, you have to carry your phone and another device, because the FDA doesn’t want glucose going through the phone. That’s really unfortunate; people would rather not pull out a glucose monitor in public. If it were in their phone, it would look like they’re just checking email.

Wired: But people with diabetes have many tools to manage their disease, and they’re self-tracking their care. You could argue that’s the epitome of digitizing medicine and giving people access to tools. They should feel empowered. But here’s what I call the diabetic’s paradox: When you survey them about these tools, they say they’re a source of frustration and anxiety—all these negative emotions. Giving them this responsibility and the tools seems to be a burden.

Topol: That’s an important issue. Will having more information induce fear and anxiety? I personally believe that if the information is easy to obtain and work with, most people would want to have it. For diabetes in particular, we know there’s a relationship between lack of glucose regulation and complications like blindness and kidney failure. So if you were diabetic and you knew that you could get your glucose in a tight, normal range just by adjusting your lifestyle, wouldn’t that be great? It could be rather seamless in your life. And you could look at your data and start to figure out what works in you: How much do exercise and certain foods help? What’s going on in your life that gets your glucose out of whack? But instead, what we have now requires finger sticks multiple times per day, the ranges are fuzzy and inexact, and the tools are horrible. As long as it doesn’t involve pain, as long as it’s simple to use out of the box, this will work. This will be better. That’s what we’re aiming for.

Wired: You write a lot about imaging—x-rays, CT scans, MRIs—and how that’s all gone digital. And that’s very much a two-edged sword, as you well know.

Topol: Absolutely. We are grossly overusing imaging in this country, and that’s really scary to me. The mass use of radiation scans is way out of line with any other place in the world. There are estimates that 2 to 3 percent of cancers in the US each year are engendered by exposure to repetitive imaging. So I present this as a shout-out to consumers. When you’re asked to have a CT scan or a nuclear scan, do you know how much radiation that involves? How many of those sorts of scans have you already had? Is it necessary? Is there an alternative? I don’t think many people know about that. We need tools that let us track our radiation exposure for ourselves, each of us. So that, I think, is an important part of how we can reboot the future of medicine.

Note to Reporters and Producers: Stephen Baylin, M.D., will present this research during a press event with Stand Up To Cancer at the Annual Meeting of the American Association for Cancer Research in Chicago’s Hyatt McCormick Conference Center at 1:00 p.m. CT, Room CC10 A/B/C. Call-in will not be available for this press event, but a Webcast will be available after the event at webcast.aacr.org.

Newswise — Experimenting with cells in culture, researchers at the Johns Hopkins Kimmel Cancer Center have breathed possible new life into two drugs once considered too toxic for human cancer treatment. The drugs, azacitidine (AZA) and decitabine (DAC), are epigenetic-targeted drugs and work to correct cancer-causing alterations that modify DNA.

The researchers said the drugs also were found to take aim at a small but dangerous subpopulation of self-renewing cells, sometimes referred to as cancer stem cells, which evade most cancer drugs and cause recurrence and spread.

In a report published in the March 20, 2012, issue of Cancer Cell, the Johns Hopkins team said their study provides evidence that low doses of the drugs tested on cell cultures cause antitumor responses in breast, lung, and colon cancers.

Conventional chemotherapy agents work by indiscriminately poisoning and killing rapidly-dividing cells, including cancer cells, by damaging cellular machinery and DNA. “In contrast, low doses of AZA and DAC may re-activate genes that stop cancer growth without causing immediate cell-killing or DNA damage,” says Stephen Baylin, M.D., Ludwig Professor of Oncology and deputy director of the Johns Hopkins Kimmel Cancer Center.

Many cancer experts had abandoned AZA and DAC for the treatment of common cancers, according to the researchers, because they are toxic to normal cells at standard high doses, and there was little research showing how they might work for cancer in general. Baylin and his colleague Cynthia Zahnow, Ph.D., decided to take another look at the drugs after low doses of the drugs showed a benefit in patients with a pre-leukemic disorder called myelodysplastic syndrome (MDS). Johns Hopkins investigators also showed benefit of low doses of the drugs in tests with a small number of advanced lung cancer patients. “This is contrary to the way we usually do things in cancer research,” says Baylin, noting that “typically, we start in the laboratory and progress to clinical trials. In this case, we saw results in clinical trials that made us go back to the laboratory to figure out how to move the therapy forward.”

For the research, Baylin and Zahnow’s team worked with leukemia, breast, and other cancer cell lines and human tumor samples using the lowest possible doses that were effective against the cancers. In all, the investigators studied six leukemia cell lines, seven leukemia patient samples, three breast cancer cell lines, seven breast tumor samples (including four samples of tumors that had spread to the lung), one lung cancer tumor sample, and one colon cancer tumor sample. The team treated cell lines and tumor cells with low-dose AZA and DAC in culture for three days and allowed the drug-treated cells to rest for a week. Treated cells and tumor samples were then transplanted into mice where the researchers observed continued antitumor responses for up to 20 weeks. This extended response was in line with observations in some MDS patients who continued to have anticancer effects long after stopping the drug.

The low-dose therapy reversed cancer cell gene pathways, including those controlling cell cycle, cell repair, cell maturation, cell differentiation, immune cell interaction, and cell death. Effects varied among individual tumor cells, but the scientists generally saw that cancer cells reverted to a more normal state and eventually died. These results were caused, in part, by alteration of the epigenetic, or chemical environment, of DNA. Epigenetic activities turn on certain genes and block others, says Zahnow, assistant professor of oncology and the Evelyn Grolman Glick Scholar at Johns Hopkins.

The research team also tested AZA and DAC’s effect on a type of metastatic breast cancer cell thought to drive cancer growth and resist standard therapies. Metastatic cells are difficult to study in standard laboratory tumor models, because they tend to break away from the original tumor and float around in blood and lymph fluids. The Johns Hopkins team re-created the metastatic stem cells’ environment, allowing them to grow as floating spheres. “These cells were growing well as spheres in suspension, but when we treated the cells with AZA, both the size and number of spheres were dramatically reduced,” says Zahnow.

The precise mechanism of how the drugs work is the focus of ongoing studies by Baylin and his team. “Our findings match evidence from recent clinical trials suggesting that the drugs shrink tumors more slowly over time as they repair altered mechanisms in cells and genes return to normal function and the cells may eventually die,” says Baylin.

The results of clinical trials in lung cancer, led by Johns Hopkins’ Charles Rudin, M.D., and published late last year in Cancer Discovery, also indicate that the drugs make tumors more responsive to standard anticancer drug treatment. This means, they say, that the drugs could become part of a combined treatment approach rather than a stand-alone therapy and as part of personalized approaches in patients whose cancers fit specific epigenetic and genetic profiles.

Low doses of both drugs are approved by the U.S. Food and Drug Administration for the treatment of MDS and chronic myelomonocytic leukemia (CMML). Clinical trials in breast and lung cancer have begun in patients with advanced disease, and trials in colon cancer are planned.

In addition to Baylin and Zahnow, other investigators participating in this study include Hsing-Chen Tsai, Huili Li, Leander Van Neste, Yi Cai, Carine Robert, Feyruz V. Rassool, James J. Shin, Kirsten M. Harbom, Robert Beaty, Emmanouil Pappou, James Harris, Ray-Whay Chiu Yen, Nita Ahuja, Malcolm V. Brock, Vered Stearns, David Feller-Kopman, Lonny B. Yarmus, Yi-Chun Lin, Alana L. Welm, Jean-Pierre Issa, Il Minn, William Matsui, Yoon-Young Jang, and Saul J. Sharkis.

The research was funded by a SPORE grant for lung cancer from the National Institutes of Health, the Hodson Trust Foundation, Entertainment Industry Foundation, Lee Jeans, Samuel Waxman Cancer Research Foundation, Department of Defense Breast Cancer Research Program, Huntsman Cancer Foundation, and the Cindy Rosencrans Fund for Triple Negative Breast Cancer Research. All of the studies have been accelerated by funding from the Stand Up to Cancer (SU2C) project in partnership with the American Association of Cancer Research (AACR).

On the Web:

Clinical trial of epigenetics therapy published in Cancer Discovery: http://www.hopkinsmedicine.org/
news/media/releases/combination_epigenetic_therapy_clinical_trial_results_

What is epigenetics?
http://www.hopkinsmedicine.org/
kimmel_cancer_center/research_clinical_trials/research/su2c/what_is_epigenetics.html

SU2C Epigenetics Dream Team: http://youtu.be/KgXBrxvlUeA

Stephen Baylin, M.D., explains epigenetics: http://youtu.be/UW3f2XAxjdM