20090812-9

Denosumab Helps Men Undergoing Prostate Cancer Treatment Avoid Fractures
GoogleNews.com, MedPageToday.com, ABCNews.com, August 11, 2009, by John Gever  —  An experimental biologic drug for osteoporosis increased bone density and reduced fractures in men and women in two controlled clinical trials.

Three years of treatment with denosumab reduced radiographic spine fractures more than two-thirds in the randomized FREEDOM study of nearly 7,900 postmenopausal women.

Overall, those fractures appeared in 2.3 percent of the denosumab group, compared with 7.2 percent of patients taking placebo, according to Dr. Steven Cummings of California Pacific Medical Center in San Francisco and colleagues.

And a separate trial by Dr. Matthew Smith of Massachusetts General Hospital in Boston and colleagues documented a similar reduction in new vertebral fractures after two years of denosumab in 1,468 men undergoing androgen-deprivation therapy for prostate cancer, and hence at risk for bone loss.

Both studies were published online in the New England Journal of Medicine. Both sets of researchers reported increases in bone mineral density (BMD) at various body sites with denosumab, whereas no change or decreases in BMD were seen in the placebo groups.

Preliminary results from the two studies were reported last year.

Sundeep Khosla, MD, of the Mayo Clinic in Rochester, Minn., in an accompanying NEJM editorial.Although the drug did not seem to increase infection rates in either study, Khosla noted that its potential to depress immune function remained an issue.

He added that its cost could “considerably limit its use” if it turns out to be much higher than zoledronic acid (Reclast), the bisphosphonate drug that appears to be denosumab’s most direct competitor.

The double-blind, randomized, placebo-controlled study by Cummings and colleagues enrolled 7,868 women 60 to 90 years old with established osteoporosis.

Patients received calcium supplements of at least 1,000 mg/day. Vitamin D supplements were provided as well. No bisphosphonates or other osteoporosis drugs were allowed.

Denosumab was given as an injection every six months for three years. Lateral spine radiographs were taken annually and analyzed at a central facility for new vertebral fractures.

New Drug Appears to Cut Fracture Risk in Men, Women

At the three-year evaluation, the researchers found that the risk for new vertebral fractures detected by imaging in the group taking denosumab are only about one-third of that in those taking placebo. Similar reductions were seen in rates of vertebral fracture cases seen by doctors and the number of cases involving two or more vertebral fractures detected by X-ray. The group receiving denosumab also enjoyed better improved density.

In the other study, involving men with prostate cancer undergoing anti-androgen therapy, the basic pattern of results was similar. As in the FREEDOM trial, the drug was given every six months by injection.

Smith and colleagues found the overall incidence of new vertebral fractures in the study after three years of treatment was 1.5 percent with denosumab versus 3.9 percent among placebo patients.

Adverse effects were largely similar between treatment groups in both studies, though the male patients showed slightly higher rates (34.6 percent versus 30.6 percent for serious events and 5.9 percent versus 4.6 percent for serious events related to infections). But notably absent from the list of adverse events was osteonecrosis of the jaw, a rare but worrisome side effect of bisphosphonate drugs.

Although the data do not rule out the possibility that osteonecrosis could occur with denosumab, it was encouraging to see no cases thus far, Cummings suggested. He said follow-up of FREEDOM participants would continue for 10 years, which may provide a more definitive view of the risk.

Denosumab’s manufacturer, Amgen, has not yet announced the pricing for the drug. Nevertheless, as a biologic drug, denosumab is widely expected to be relatively expensive.

But another academic specialist contacted by MedPage Today and ABC News suggested a higher price could be acceptable given denosumab’s potential advantages.

Endocrinologist Dr. Roberto Pacifici of Emory University in Atlanta said the “number needed to treat” in preventing fractures has been low in the denosumab studies reported to date.

The Question of Cost

“Therefore the cost of the drug is likely to be [worthwhile] in many patients,” he said. “Guidelines will have to be developed in order to use this agent in the most cost-effective way.”

Cummings said that if the drug is approved, he would consider it primarily for patients with a poor history on bisphosphonates.

“Those who have had trouble with oral drugs,” because of side effects or compliance problems, would be the main candidates for denosumab, he said.

Another point in denosumab’s favor, Cummings said, is that it is less persistent in bone than many bisphosphonates, making its activity potentially reversible  an important point for patients experiencing adverse effects.

In addition, Dr. Khosla pointed out, “since bisphosphonates are cleared by the kidney and contraindicated in patients with renal insufficiency, denosumab (which is cleared by nonrenal metabolism) may prove to be a safe drug in these patients, although studies that directly address this issue need to be done.”

Amgen filed last December for FDA approval of the agent for treatment and prevention of postmenopausal osteoporosis in women and treatment and prevention of bone loss in patients undergoing hormone ablation therapy for either prostate or breast cancer. The two trials were funded by Amgen.

This article was developed in collaboration with ABC News.

20090812-7

GoogleNews.com, August 11, 2009 (PRNews)  Twice-Yearly Administration of Denosumab Resulted in 68 Percent Reduction in Risk for a Vertebral Fracture and 40 Percent Reduction in Risk for a Hip Fracture in Women with Postmenopausal Osteoporosis

Denosumab Administered Twice-Yearly Reduced the Incidence of New Vertebral Fractures by 62 Percent in Men with Non-Metastatic Prostate Cancer Undergoing Androgen Deprivation Therapy

THOUSAND OAKS, Calif., Aug. 11 /PRNewswire-FirstCall/ — Amgen Inc. (Nasdaq: AMGN) today announced the publication of results from two pivotal Phase 3 studies investigating the safety and effectiveness of denosumab at reducing fracture risk in more than 7,800 women with postmenopausal osteoporosis and in more than 1,400 men with non-metastatic prostate cancer undergoing androgen deprivation therapy (ADT) leading to bone loss. In both studies, published today in The New England Journal of Medicine (NEJM), patients receiving twice-yearly denosumab experienced significant increases in bone mineral density (BMD) compared to placebo, associated with more than 60 percent reduction in vertebral fracture in both patient populations.(1,2) These data were previously reported by Amgen at medical congresses.

To view the Multimedia News Release, go to:http://www.prnewswire.com/mnr/amgen/39203/

“The discovery of the RANK Ligand pathway represents a significant advance in the understanding of bone biology,” said Roland Baron, Ph.D., D.D.S., professor and chair of department of Oral Medicine, Infection, and Immunity at the Harvard School of Dental Medicine. “These results demonstrate that targeting the RANK Ligand pathway with denosumab could represent a promising new approach in two different disease settings characterized by bone loss.”

FREEDOM Osteoporosis Study Results: Significant Fracture Reduction Seen Across the Skeleton in Postmenopausal Women with Osteoporosis

Results from the FREEDOM (Fracture REduction Evaluation of Denosumab in Osteoporosis every six Months) study, showed that women receiving a subcutaneous shot of denosumab twice-yearly experienced a 68 percent reduction in the risk of suffering a vertebral (spine) fracture compared to those receiving placebo as well as a 40 percent reduction in the risk of suffering a hip fracture and a 20 percent reduction in the risk of suffering a nonvertebral fracture. Over the three years of this multi-center, randomized, double-blind, placebo-controlled study, women treated with denosumab experienced significant increases in BMD (8.8 percent at the lumbar spine and 6.4 percent at the total hip).(1)

“These results suggest that denosumab offers a new approach to prevention of fractures in women with postmenopausal osteoporosis,” said Steven Cummings M.D., lead investigator, study author, and director of the San Francisco Coordinating Center of the California Pacific Medical Center Research Institute. “It reduces the risk of all major types of fractures and, because it is given as an injection twice a year, it also has the potential to help compliance to treatment.”

Fracture is one of the most common health events suffered by postmenopausal women with osteoporosis.(3) Globally, one woman in three over 50 years of age will experience a fracture in her lifetime.(3) A woman who has broken a bone as a result of osteoporosis has more than an eight- out-of-ten chance of breaking another bone.(4) Half of women who break a hip, a life changing event, will permanently need assistance to walk.(5)

The overall incidence and type of side effects with denosumab were similar to placebo in the FREEDOM study. Rates of adverse events (AEs) were similar in both groups (93 percent). Rates of serious AEs were 25.8 percent for denosumab and 25.1 percent for placebo. The most common AEs across both treatment arms were arthralgia, back pain, hypertension and nasopharyngitis. There were no reported cases of osteonecrosis of the jaw among patients taking denosumab. Serious adverse events of skin infections, predominantly cellulitis, were reported more commonly in the denosumab group (0.4 percent vs. <0.1 percent). Mild, transient decreases in serum calcium were observed that had no apparent clinical significance.(1)

HALT Study Results: First Published Study to Demonstrate Fracture Prevention in Men with Non-Metastatic Prostate Cancer Undergoing ADT

Results from the HALT (Hormone AbLation Therapy) study in 1,468 men undergoing ADT for non-metastatic prostate cancer show that patients treated with denosumab experienced a 62 percent reduction in the risk of suffering a new vertebral fracture with denosumab compared to placebo at 36 months, with significant reduction observed as early as month-12.(2) Bone loss and increased fracture risk are serious and under-recognized consequences of ADT(6,7) and currently there are no approved therapies for these patients.

In this multi-center, randomized, double-blind, placebo-controlled study, men receiving 60 mg denosumab administered subcutaneously experienced a 6.7 percent increase in BMD at the lumbar spine compared to those receiving placebo (primary endpoint) at 24 months. Increases in BMD at the lumbar spine were observed as early as one month after starting treatment with denosumab and continued to increase throughout the study. In addition, denosumab produced significant increases in BMD at non-vertebral sites (total hip 4.8 percent, femoral neck 3.9 percent, and distal 1/3 radius 5.5 percent), compared to placebo.(2)

“Bone loss and fractures are an important but often unrecognized problem for prostate cancer survivors. Bone loss is an early adverse effect and even short-term androgen deprivation therapy negatively impacts skeletal health. Prevention of bone loss and fractures has been a key unmet medical need for men with prostate cancer,” said Matthew Smith, M.D., Ph.D., study author, associate professor of medicine and the director of Genitourinary Medical Oncology at Massachusetts General Hospital Cancer Center. “In this large international study, denosumab markedly increased bone mineral density and decreased the risk of fractures in many men receiving androgen deprivation therapy for prostate cancer. The efficacy of denosumab was apparent as early as one month and was sustained for three years.”

In the HALT trial, the overall incidence and type of side effects with denosumab were similar to placebo. Rates of AEs were similar in both groups (87 percent). Rates of serious AEs were 35 percent for denosumab and 31 percent for placebo. The most common AEs across both treatment arms were arthralgia, back pain, constipation, pain in extremity, and hypertension. There were no reported cases of osteonecrosis of the jaw among patients treated with denosumab. More patients receiving denosumab developed cataracts, though none were considered treatment-related. One patient in the denosumab arm developed hypocalcemia, versus none in the placebo arm. New primary malignancies were reported in 5 percent of patients in each group. Serious AEs of infections were reported in 6 percent of denosumab-treated patients and in 5 percent of placebo-treated patients.(2)

“Amgen scientists in the 1990s were the first to identify the RANK Ligand pathway, a pivotal physiologic mechanism that controls bone remodeling,” said Roger Perlmutter, M.D., Ph.D., executive vice president of Research and Development at Amgen. “Today’s publications in the New England Journal of Medicine underscore the significance of this finding, and highlight Amgen’s focus on using innovative research to address grievous illness.”

About Denosumab

Denosumab is the first fully human monoclonal antibody in late stage clinical development that specifically targets RANK Ligand, an essential regulator of osteoclasts (the cells that break down bone). Denosumab is being investigated for its potential to inhibit all stages of osteoclast activity through a targeted mechanism. Denosumab is being studied in a range of bone loss conditions including postmenopausal osteoporosis and bone loss in patients undergoing hormone ablation for prostate and breast cancer.

In February 2009, the U.S. Food and Drug Administration (FDA) accepted the Biologics License Application (BLA), submitted by Amgen for denosumab for the treatment and prevention of osteoporosis in postmenopausal women and cancer treatment-induced bone loss in women and men receiving hormone therapy for either breast cancer or prostate cancer based on these studies and a parallel trial in women with breast cancer. The FDA has provisionally approved the trade name Prolia(TM) in these proposed indications, for which denosumab is administered twice yearly subcutaneously at a 60mg dose. The trade name is only for these indications and may not apply for other indications of denosumab.

Amgen has also submitted marketing applications for use of denosumab in the European Union, Canada, Switzerland, and Australia.

Osteoporosis: Impact and Prevalence

Often referred to as the “silent epidemic,” osteoporosis is a global problem that is increasing in significance as the population of the world both increases and ages. The World Health Organization (WHO) has recently identified osteoporosis as a priority health issue along with other major non-communicable diseases.

The economic burden of osteoporosis is comparable to that of other major chronic diseases; for example, in the U.S., the costs associated with osteoporosis-related fractures are equivalent to those of cardiovascular disease and asthma.(8,9,10) It has been reported that osteoporosis results in more hospital bed-days than stroke, myocardial infarction or breast cancer.(11)

Bone Loss Due to Hormone Ablation Therapy

Worldwide, prostate cancer and breast cancer are two of the most frequent types of cancer affecting men and women, respectively.(12) In the U.S., prostate cancer is the most common cancer in men and breast cancer is the most common cancer in women. It is common for prostate cancer and breast cancer patients to receive hormone ablation therapies that can lead to a decrease in bone mass and increased risk of fractures. Currently there are no approved therapies for bone loss in patients undergoing hormone ablation for either prostate or breast cancer.

About Amgen

Amgen discovers, develops, manufactures and delivers innovative human therapeutics. A biotechnology pioneer since 1980, Amgen was one of the first companies to realize the new science’s promise by bringing safe and effective medicines from lab, to manufacturing plant, to patient. Amgen therapeutics have changed the practice of medicine, helping millions of people around the world in the fight against cancer, kidney disease, rheumatoid arthritis, and other serious illnesses. With a deep and broad pipeline of potential new medicines, Amgen remains committed to advancing science to dramatically improve people’s lives. To learn more about our pioneering science and our vital medicines, visit www.amgen.com.

Forward-Looking Statements

This news release contains forward-looking statements that are based on management’s current expectations and beliefs and are subject to a number of risks, uncertainties and assumptions that could cause actual results to differ materially from those described. All statements, other than statements of historical fact, are statements that could be deemed forward-looking statements, including estimates of revenues, operating margins, capital expenditures, cash, other financial metrics, expected legal, arbitration, political, regulatory or clinical results or practices, customer and prescriber patterns or practices, reimbursement activities and outcomes and other such estimates and results. Forward-looking statements involve significant risks and uncertainties, including those discussed below and more fully described in the Securities and Exchange Commission (SEC) reports filed by Amgen, including Amgen’s most recent annual report on Form 10-K and most recent periodic reports on Form 10-Q and Form 8-K. Please refer to Amgen’s most recent Forms 10-K, 10-Q and 8-K for additional information on the uncertainties and risk factors related to our business. Unless otherwise noted, Amgen is providing this information as of Aug. 11, 2009, and expressly disclaims any duty to update information contained in this news release.

No forward-looking statement can be guaranteed and actual results may differ materially from those we project. Discovery or identification of new product candidates or development of new indications for existing products cannot be guaranteed and movement from concept to product is uncertain; consequently, there can be no guarantee that any particular product candidate or development of a new indication for an existing product will be successful and become a commercial product. Further, preclinical results do not guarantee safe and effective performance of product candidates in humans. The complexity of the human body cannot be perfectly, or sometimes, even adequately modeled by computer or cell culture systems or animal models. The length of time that it takes for us to complete clinical trials and obtain regulatory approval for product marketing has in the past varied and we expect similar variability in the future. We develop product candidates internally and through licensing collaborations, partnerships and joint ventures. Product candidates that are derived from relationships may be subject to disputes between the parties or may prove to be not as effective or as safe as we may have believed at the time of entering into such relationship. Also, we or others could identify safety, side effects or manufacturing problems with our products after they are on the market. Our business may be impacted by government investigations, litigation and products liability claims. We depend on third parties for a significant portion of our manufacturing capacity for the supply of certain of our current and future products and limits on supply may constrain sales of certain of our current products and product candidate development.

In addition, sales of our products are affected by the reimbursement policies imposed by third-party payors, including governments, private insurance plans and managed care providers and may be affected by regulatory, clinical and guideline developments and domestic and international trends toward managed care and healthcare cost containment as well as U.S. legislation affecting pharmaceutical pricing and reimbursement. Government and others’ regulations and reimbursement policies may affect the development, usage and pricing of our products. In addition, we compete with other companies with respect to some of our marketed products as well as for the discovery and development of new products. We believe that some of our newer products, product candidates or new indications for existing products, may face competition when and as they are approved and marketed. Our products may compete against products that have lower prices, established reimbursement, superior performance, are easier to administer, or that are otherwise competitive with our products. In addition, while we routinely obtain patents for our products and technology, the protection offered by our patents and patent applications may be challenged, invalidated or circumvented by our competitors and there can be no guarantee of our ability to obtain or maintain patent protection for our products or product candidates. We cannot guarantee that we will be able to produce commercially successful products or maintain the commercial success of our existing products. Our stock price may be affected by actual or perceived market opportunity, competitive position, and success or failure of our products or product candidates. Further, the discovery of significant problems with a product similar to one of our products that implicate an entire class of products could have a material adverse effect on sales of the affected products and on our business and results of operations.

The scientific information discussed in this news release related to our product candidates is preliminary and investigative. Such product candidates are not approved by the U.S. Food and Drug Administration (FDA), and no conclusions can or should be drawn regarding the safety or effectiveness of the product candidates. Only the FDA can determine whether the product candidates are safe and effective for the use(s) being investigated. Further, the scientific information discussed in this news release relating to new indications for our products is preliminary and investigative and is not part of the labeling approved by the U.S. Food and Drug Administration (FDA) for the products. The products are not approved for the investigational use(s) discussed in this news release, and no conclusions can or should be drawn regarding the safety or effectiveness of the products for these uses. Only the FDA can determine whether the products are safe and effective for these uses. Healthcare professionals should refer to and rely upon the FDA-approved labeling for the products, and not the information discussed in this news release.

    CONTACT: Amgen, Thousand Oaks

    Sarah Reines:  (805) 447-9783  (media, osteoporosis)

    Lisa Rooney:  (805) 447-6437  (media, oncology)

    Arvind Sood:  (805) 447-1060  (investors)

(Logo: http://www.newscom.com/cgi-bin/prnh/20081015/AMGENLOGO)

References

  1. Cummings SR, et al. Twice Yearly Denosumab, a Monoclonal Antibody to RANK-ligand, for Prevention of Fractures in Postmenopausal Women with Osteoporosis. N Engl J Med, 2009 Aug. 20; published online at www.nejm.org on Aug. 11, 2009.
  2. Smith MR, et al. Denosumab for the Prevention of Bone Loss and Fractures in Men Receiving Androgen Deprivation Therapy in Non-Metastatic Prostate Cancer. N Engl J Med, 2009 Aug. 20; published online at www.nejm.org on Aug. 11, 2009.
  3. Melton LJ, et al. (1992) Perspective. How Many Women Have Osteoporosis? J Bone Miner Res, 1992;7:1005
  4. Kanis JA, et al. A Meta-Analysis of Previous Fracture and Subsequent Fracture Risk. Bone, 2004;35:375.
  5. Magaziner J, et al. Predictors of Functional Recovery One Year Following Hospital Discharge for Hip Fracture: A Prospective Study. J Gerontol, 1990;45:M101.
  6. Higano 2008; Higano 2004; Conde 2003; Smith 2001; Pfeilschifter 2000.
  7. Oefelein MG, Ricchiuti V, Conrad W, Resnick MI. Skeletal fractures negatively correlate with overall survival in men with prostate cancer. J Urol. 2002;168:1005-1007.
  8. Burge R, et al. J Bone Miner Res. 2007; 22:465-475
  9. “Osteoporosis Fast Facts.” Washington (DC): National Osteoporosis Foundation. Accessed on February 24, 2009 at http://www.nof.org/osteoporosis/stats.html.
  10. “Economic Cost of Cardiovascular Diseases.” Dallas (TX): American Heart Association. Accessed on February 24, 2009 at http://www.americanheart.org/statistics/10econom.html.
  11. Lippuner K, et al. “Incidence and direct medical costs of hospitalisations due to osteoporotic fractures in switzerland.” Osteoporosis International.1997;7:414-25.
  12. WHO. Media Center. Fact sheet No. 297. February 2009 at http://www.who.int/mediacentre/factsheets/fs297/en/index.html

Source: Amgen Inc.

20090812-2

One step closer to personalized medicine

Stanford University Medical Center (2009, August 11). Professor Sequences His Entire Genome At Low Cost, With Small Team, ScienceDaily  –  The first few times that scientists mapped out all the DNA in a human being in 2001, each effort cost hundreds of millions of dollars and involved more than 250 people. Even last year, when the lowest reported cost was $250,000, genome sequencing still required almost 200 people. 

In a paper published online Aug. 9 by Nature Biotechnology, a Stanford University professor reports sequencing his entire genome for less than $50,000 and with a team of just two other people. 

In other words, a task that used to cost as much as a Boeing 747 airplane and required a team of people that would fill half the plane, now costs as much as a mid-priced luxury sedan and the personnel would fill only half of that car.

“This is the first demonstration that you don’t need a genome center to sequence a human genome,” said Stephen Quake, PhD, professor of bioengineering. “It’s really democratizing the fruits of the genome revolution and saying that anybody can play in this game.”

There are at least two reasons why lowering the cost and effort required to sequence all the genetic information of individuals is important. The more examples scientists have of the whole human genetic code, the more they can discern about how specific genes and mutations result in the traits that make us all different, the diseases that plague us and our response to medicines. As that understanding increases and costs drop, doctors could then sequence their patients’ genomes and provide “personalized medicine” in which prevention and treatment of disease would be informed by the patient’s exact genetic profile.

“This can now be done in one lab, with one machine, at a modest cost,” said Quake, the Lee Otterson Professor in the School of Engineering and a member of Stanford’s Cancer Center. “It’s going to unleash an enormous amount of creativity and really broaden the field.”

Quake’s genome, one of less than a dozen sequenced so far because of the cost and resources needed, is now available to researchers worldwide. Quake’s colleagues at Stanford’s School of Medicine have been looking through it and sometimes examining Quake himself, mining the data for interesting connections between what they can observe about him, his DNA and his family history.
 
“Some of the doctors are starting to poke and prod me to see how they can couple my genome with medicine,” he said.

Simpler sequencing

To sequence his genome, Quake’s team used a commercially available, refrigerator-sized instrument called the Helicos Biosciences SMS Heliscope. Quake, who pioneered the underlying technology in 2003, is a co-founder of the Cambridge, Mass.-based company and chairs its scientific advisory board.
 

The technology-the SMS in the instrument’s name-is called single molecule sequencing. While many techniques require generating thousands of copies of a subject’s DNA, the single molecule technique does not, reducing the cost and effort involved. Instead, the technique requires chopping the 3 billion or so fundamental units of DNA (called bases) into strands about 30 bases long. The four bases in DNA are adenine (abbreviated A), cytosine (C), guanine (G), and thymine (T).
 

Each base of DNA matches with a specific other base: For example, T only matches with A. The machine captures each of the millions of strands on a specially treated glass plate, holds them there and washes successive waves of fluorescently labeled “letters” over the plate. As each complementary letter sticks next to a strand, the machine can read out the sequence of each strand. A video of the process can be seen on the Web: http://www.helicosbio.com/Technology/TrueSingleMoleculeSequencing/tabid/64/Default.aspx.

Assembling the strands back into a cohesive genome is then done by powerful computers, which compare it to the reference genomes that have been compiled before. The process is akin to assembling an enormous jigsaw puzzle by referring frequently to the picture on the box. The team said the sequencing process took about one month to complete.

Still, several tricky problems had to be solved before the machine could reliably sequence a whole human genome. Quake worked with Norma Neff, a research manager in Quake’s lab, and physics doctoral student Dmitry Pushkarev to write a sophisticated algorithm that would enable them to determine how accurate the process is.

Overall, the genome is 95 percent complete, a rate comparable with other sequenced genomes, the team found. In the paper, the authors are careful to note that all genome-sequencing technologies, including the one they’ve demonstrated, have produced incomplete approximations of the actual genome. Still, it is enough to help produce genuine insights about a person’s traits and health.

A professor’s personal revelations

Quake’s genome has already yielded a few interesting connections between his genetics and his health. One is that he carries a rare mutation associated with a heart disorder; the revelation, he said, sheds light on what members of his family have always wondered with regard to the health of prior generations. The good news, he said, is that he’s also apparently genetically predisposed to respond well to common cholesterol-lowering statin medicines.

Quake said the information has also forced him to take heed of that history. “If you know your uncle had something, you kind of discount that you can get it, but to see you’ve inherited the mutation for that is another matter altogether,” he said.

One amusing “revelation” is that Quake’s code contains a form of a gene that has sometimes been associated with increased disagreeability, he said. The details of the code can be found on the Web at http://www.snpedia.com/index.php/Rs6832769.

“Of course, you don’t need my genome to tell you that,” Quake acknowledged. “My wife could have told you that and certainly the dean could have as well.”
 

Funding for the research came from the National Science Foundation and from the National Institutes of Health.


 

Adapted from materials provided by Stanford University Medical Center. Original article written by David Orenstein, associate communications director, School of Engineering.

20090812-3

True Single Molecule Sequencing

By directly sequencing single molecules of DNA or RNA, Helicos’ True Single Molecule Sequencing(tSMS)TM technology significantly increases the speed of sequencing, while also decreasing the cost.

tSMS enables the simultaneous sequencing of large numbers of strands of single DNA or RNA molecules by using a proprietary form of sequencing-by-synthesis in which labeled DNA bases are sequentially added to the nucleic acid templates captured on a flow cell.  Our optimized formulation ensures high accuracy of each base addition, which are detected by the HeliScopeTM Single Molecule Sequencer to elucidate the sequence of bound strands.

Download the Helicos tSMS Technology Primer (pdf). 

How tSMS Works

Within two flow cells, billions of single molecules of sample DNA are captured on an application-specific proprietary surface. These captured strands serve as templates for the sequencing-by-synthesis process:

Polymerase and one fluorescently labeled nucleotide (C, G, A or T) are added.

  • The polymerase catalyzes the sequence-specific incorporation of fluorescent nucleotides into nascent complementary strands on all the templates.
  • After a wash step, which removes all free nucleotides, the incorporated nucleotides are imaged and their positions recorded.
  • The fluorescent group is removed in a highly efficient cleavage process, leaving behind the incorporated nucleotide.
  • The process continues through each of the other three bases.
  • Multiple four-base cycles result in complementary strands greater than 25 bases in length synthesized on billions of templates-providing a greater than 25-base read from each of those individual templates.

 

Raw tSMS Image

20090812-4

An image taken by the HeliScope Single Molecule Sequencer.
Inset shows a close-up view of individual single molecules.

Watch the Video
“How True Single Molecule Sequencing Works”

http://www.helicosbio.com/Technology/TrueSingleMoleculeSequencing/tabid/64/Default.aspx

The Data Pipeline

The data pipeline of the HeliScopeTM Single Molecule Sequencer is designed for openness and flexibility. Data is available for export at several points during the production process.

Images, the object table, and sequence, trace and quality data are output in non-proprietary formats, either through the HeliScope Web Interface or HeliScope Application Programming Interfaces (APIs). We also use the HeliScope APIs in our internal development, ensuring quality of performance and timely maintenance.

20090812-5

Image Analysis

Using the latest in high-performance-computing technology and state of the art optics, the system’s image analysis software, located on the HeliScopeTM Analysis Engine, identifies and extracts millions of nucleotide base incorporations from the 48 images produced by the HeliScopeTM Single Molecule Sequencer each second – in near real time.

Features are identified according to stringent nucleotide incorporation criteria and catalogued in an “object table” for subsequent base calling and sequence formation.

20090812-6

True Biology

 

Direct Measurement: The Pathway to True Biology
Limitations in the detection sensitivity of traditional biological analysis tools, have often forced scientists to measure the properties of molecular aggregates and assume that these properties represent a true characterization of the constituent individual molecules. An example is high-throughput nucleic acid sequencing. Many current sequencing approaches depend on an amplification step to copy DNA and RNA before the base sequence is determined. Sequencing the copies of the sample, rather than the sample itself can mask the biology of the actual sample by introducing biases and errors.
 
Measure the biology of the cell; not the biology of the test tube
The essence of true biology is the ability to interrogate individual molecules of a sample directly, without depending on proxies. Helicos True Single Molecule Sequencing (tSMS)TM technology captures exactly that ability – to sequence and detect individual nucleic acid molecules without relying on an amplification process. With single molecule sequencing technology, the Helicos genetic analysis tools are poised to usher in a new era of high-fidelity, high-throughput genomic exploration.

True Direct DNA measurement enables high-resolution science

  • Accurate – The ability to optically detect individual nucleic acid incorporation events on a single strand of DNA or RNA allows unparalleled sequence accuracy.
  • Simple – Sample preparation is minimal, and does not require an amplification process. Instrument interface is intuitive and instrument operation is automated.
  • Unbiased – PCR-based DNA amplification can lead to biases because certain fragments reproduce at different rates, leading to the systematic overrepresentation of some segments and an underrepresentation of others. Helicos tSMS technology allows amplification-free sample prep.
  • Scalable – The density allowed by single molecule sequencing along with the flow cell design allows excellent scalability of samples and target size.
  • Sensitive – Single molecule detection and minimally altered samples allows unprecedented looks at rare mutations and transcripts expressed at low levels.

Helicos Translates Single Molecule Sequencing Into Real Research Power

  • Greater quantitative power to obtain true counts, rather than ratiometric data
  • Application flexibility for Digital Gene Expression, Targeted Resequencing, Copy-Number Variation Assessment, Chromatin Immunoprecipitation-Sequencing and others
  • Enhanced ability to discern rare mutations in heterogeneous samples and measure rare transcripts
  • Demonstrated freedom from amplification and fragment-length bias as well as from sequence context effects
  • Generation of more data through single molecule template density and flow cell design

Scalability and Simplicity

Bringing Together Power and Practicality
The door to true biology has been opened by the ability of the HelicosTM Genetic Analysis System to directly sequence and image individual molecules. The power of the single molecule approach can only be fully realized by integrating onto a robust and versatile instrument design and combining it with easy sample preparation. Helicos recognizes this, and has coupled the power of single molecule sequencing with the practicality of a flexible, easy-to-use instrument and simple sample preparation.  Detection at the single molecule level is only the beginning!

Scalability by design
Powered by Helicos tSMSTM technology, the system generates more data per run of any available sequencing technique, providing unmatched scalability. Superior data output is a result of the high density of sequenced DNA strands on the flow cell surface, as well as the 25-channel flow cell design (50 channels total). Generating this amount of data per run translates into a wealth of experimental flexibility, and into increased experimental validity.

Whether you have only tiny amounts of precious or degraded samples, or a large, genome-scale sample, the Helicos Genetic Analysis System offers:

  • Breadth of coverage-Run up to 50 samples simultaneously for deep coverage across a wide area or the whole genome, including both AT-rich and CG-rich regions
  • Depth of coverage-Run one sample on multiple channels simultaneously for deep coverage of a specific region
  • Elimination of variation-Increase data reliability and validity by avoiding run-to-run variation, operator-to-operator variation and reagent, sample loading or hybridization variability

The scalability enabled by the technology coupled with the accuracy of single molecule sequencing means experiments with high sensitivity.

Simplicity Start to Finish:
Helicos is dedicated to enabling your success.  So we combine the ability to conduct a range of different experiments on a single platform with the simplest sample preparation.

  1. Fewer steps saves time and money-fragment DNA, tail and hybridize to surface
  2. No amplification-single molecule sequencing does not require additional amplification

Intuitive instrument operation-the instrument has an easy-to-use touch screen interface and has fully automated reagent delivery and imaging capability

Genetic Tests Advertised Directly To The Consumer

SAGE Publications (2009, August 11). Genetic Tests Advertised Directly To The Consumer, ScienceDaily  –  Genetic testing services have recently begun to be advertised directly to the patient, and the results of the consumers’ response can affect public health, as well as the future adoption of pharmacogenetic/genomic testing, according to a position paper from the American College of Clinical Pharmacology (ACCP) to be published in the August 2009 issue of the Journal of Clinical Pharmacology. 

According to the article prescription medications have been advertised in the US for 10+ years with a defined oversight of the process, however, there’s no comparable supervision for advertised genetic testing. In order for the consumer to have a positive experience with choosing genetic testing from advertising, clinical pharmacologists believe that effective governmental oversight is imperative, along with assistance from professional clinicians. 

Clinical pharmacologists should also help prepare consumers for possible risks of the tests and work closely with those who are considering taking further action in response to an advertisement of genetic testing, providing the following cautionary advice:

  • * Verify with a professional, the information presented in advertisements of genetic testing
  • * Seek advice from a genetic clinician, not just a general health care professional
  • * Recognize the scientific limitations of each test, especially since the information isn’t necessarily translatable when it’s time to make therapeutic decisions.
  • * Realize that many companies selling genetic testing services do not provide interpretation of results.

Commenting on the expanding frontiers of health professions, the authors conclude, “Clinical pharmacology will have a voice in how the evolving science translates not only into clinical trials and patient care but also into regulations involving the promotion and advertising of all genetic tests. Clinical pharmacologists can help ensure that realistic expectations of genetic tests are communicated, thereby mitigating psychological, social, and medical risks.”


Journal reference:

  • 1. Barbara Ameer and Norberto Krivoy. Direct-to-Consumer/Patient Advertising of Genetic Testing: A Position Statement of the American College of Clinical Pharmacology. Journal of Clinical Pharmacology, August, 2009 [link]

20090812-1

University of California – San Diego (2009, August 11). Future Of Personalized Cancer Treatment: New System Delivers RNA Into Cells, ScienceDaily.  –  In technology that promises to one day allow drug delivery to be tailored to an individual patient and a particular cancer tumor, researchers at the University of California, San Diego School of Medicine, have developed an efficient system for delivering siRNA into primary cells. The work is published in the May 17 in the advance online edition of Nature Biotechnology. 

“RNAi has an unbelievable potential to manage cancer and treat it,” said Steven Dowdy, PhD, Howard Hughes Medical Institute Investigator and professor of cellular and molecular medicine at UC San Diego School of Medicine. “While there’s still a long way to go, we have successfully developed a technology that allows for siRNA drug delivery into the entire population of cells, both primary and tumor-causing, without being toxic to the cells.”

 

For many years, Dowdy has studied the cancer therapy potential of RNA inhibition which can be used to silence genes through short interfering, double-stranded RNA fragments called siRNAs. But delivery of siRNAs has proven difficult due to their size and negative electrical charge – which prohibits them from readily entering cells. 

A small section of protein called a peptide transduction domain (PTD) has the ability to permeate cell membranes. Dowdy and colleagues saw the potential for PTDs as a delivery mechanism for getting siRNAs into cancer cells. He and his team had previously generated more than 50 “fusion proteins” using PTDs linked to tumor-suppressor proteins. 

“Simply adding the siRNAs to a PTD didn’t work, because siRNAs are highly negatively charged, while PTDs are positively charged, which results in aggregation with no cellular delivery,” Dowdy explained. The team solved the problem by making a PTD fusion protein with a double-stranded RNA-binding domain, termed PTD-DRBD, which masks the siRNA’s negative charge. This allows the resultant fusion protein to enter the cell and deliver the siRNA into the cytoplasm where it specifically targets mRNAs from cancer-promoting genes and silences them. 

To determine the ability of this PTD-DRBD fusion protein to deliver siRNA, the researchers generated a human lung cancer reporter cell line. Using green and fluorescent protein and analyzing the cells using flow cytometry analysis, they were able to determine the magnitude of RNA inhibitory response and the percentage of cells undergoing this response. They found that the entire cellular population underwent a maximum RNAi response. Similar results were obtained in primary cells and cancer cell lines. 

“We were subsequently able to introduce gene silencing proteins into a large percentage of various cell types, including T cells, endothelial cells and human embryonic stem cells,” said Dowdy. “Importantly, we observed no toxicity to the cells or innate immune responses, and a minimal number of transcriptional off-target changes.” 

These RNAi methods can be continually tweaked to combat new mutations – a way to overcome a major problem associated with current cancer therapies. “Such therapies can’t be used a second time if a cancer tumor returns, because the tumor has mutated the target gene to avoid the drug binding,” said Dowdy. “But since the synthetic siRNA is designed to bind to a single mutation and only that mutation on the genome, it can be easily and rapidly changed while maintaining the delivery system – the PTD-DRBD fusion protein.”

“Cancer is a complex, genetic disease that is different in every patient,” Dowdy added. “This is still in early stages, but I believe the siRNA-induced RNAi approach to personalized cancer treatment is the only thing on the table.” 

Additional contributors to the paper include Akiko Eguchi, Bryan R. Meade, Yung-Chi Chang, Craig T. Fredrickson, and Karl Willert of the UC San Diego School of Medicine; and Nitin Puti, Life Technologies, Austin, Texas.

The work was supported by the Howard Hughes Medical Institute, the Leukemia and Lymphoma Society, the Elsa U. Pardee Foundation, and the California Institute for Regenerative Medicine.

Dowdy is the scientific founder of Traversa Therapeutics located in La Jolla, CA, that has licensed the exclusive rights to commercialize this technology.


 

Journal reference:

•1.                 Akiko Eguchi, Bryan R Meade, Yung-Chi Chang, Craig T Fredrickson, Karl Willert, Nitin Puri & Steven F Dowdy. Efficient siRNA delivery into primary cells by a peptide transduction domain-dsRNA binding domain fusion protein. Nature Biotechnology, 2009; DOI: 10.1038/nbt.1541

Adapted from materials provided by University of California – San Diego, via EurekAlert!, a service of

Novel Sputum Tests May Help Detect Lung Cancer Early

Medscape.com, August 7, 2009, by Barbara Boughton (San Francisco, California) – Early detection of lung cancer remains one of the challenges of modern medicine. One method for early detection – sputum analysis – is plagued by inconsistent results and a high false-negative rate. Two new tests for lung cancer cells in sputum aim to improve the sensitivity and specificity associated with slide-based review, according to research presented here at the 13th World Conference on Lung Cancer, organized by the International Association for the Study of Lung Cancer (IASLC).

One of the testing methods is a device that produces 3D images of cells in sputum and distinguishes normal from abnormal cells on the basis of a variety of morphology characteristics. The other test analyzes sputum cells for 3 methylation markers that are signals of lung cancer.

The 3D test, called the Lung Cell Evaluation Device (LuCED), developed by VisionGate, uses a process that dissolves mucous in sputum, making it easier to work with the remaining cells. Cells are collected in clusters, imaged in 3D, and analyzed for more than 25 different features that can indicate how abnormal they are. These features include cell volume, cell shape, nuclear/cytoplasmic volume, and nuclear or chromatin distribution. The device then assigns a score to each specimen of cells, with higher scores indicating dysplasia or cancer. About 1000 cells are processed to obtain each score, said researcher Michael Meyer, MS, vice president for image engineering at VisionGate.

In collaboration with the University of Washington, VisionGate researchers analyzed sputum samples from more than 400 patients with confirmed lung cancer. They found that their technology had 100% specificity and 90% sensitivity, Mr. Meyer said. “This is an extremely valuable technique that could be used in many different applications. Our goal [is] to provide critical information to clinicians for follow-up,” he added. Plans call for LuCED to be commercialized for lung cancer testing by 2011. By that time, researchers estimate that the technology will be able to process each specimen in about 20 minutes; it now takes about a day.

“The sensitivity of the test is very high,” noted former IASLC board member Masahiro Fukuoka, MD, PhD, from Kinki University School of Medicine in Osaka, Japan. “This 3D analysis could provide high sensitivity and consistency in diagnosis of dysplastic and cancer cells. It could be a new diagnostic standard,” he added.

In other research presented at the meeting, Dutch scientists explored the relation between methyl groups in sputum and lung cancer, which could be used to devise a noninvasive and more accurate diagnostic test. The researchers selected 3 promising marker genes that could signal the presence of lung cancer – RASSF1A, CYGB, and APC – and examined sputum samples of 570 lung cancer patients for these markers. They found that using the 3 methylation markers together, their tests had 60% sensitivity and 90% specificity. When tests for RASSF1A and conventional cytology were combined, their tests had 54% sensitivity and 98% specificity.

“Sputum methylation was detected even in sputum samples from patients with lower-stage lung cancers,” said lead researchers Erik Thunnissen, MD, from Pathologisch Anatomisch Laboratorium in Amsterdam, the Netherlands.

Although acknowledging the superior sensitivity and specificity of the Dutch researchers’ methylation testing, Stephen Lam, MD, from the British Columbia Cancer Agency in Vancouver, cautioned that these tests may be a long way from being practical for lung cancer screening. Dr. Lam, who moderated the session at which the Dutch paper was presented, noted that the samples being analyzed came from patients with clinically diagnosed lung cancer, and that the tests could perform differently in a screening setting.

“We need to look at the incremental value of these biomarkers and integrate clinical data with this kind of analysis. In that way, biomarker tests are likely to be improved further,” he said.

Mr. Meyer is an employee of VisionGate. Dr. Fukuoka, Dr. Thunnissen, and Dr. Lam have disclosed no relevant financial relationships.

13th World Conference on Lung Cancer (WCLC): Abstracts D1.3 and D1.5. Presented August 4, 2009.