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Researchers Develop Rapid, Cost-effective Early Detection Method for Organ Transplant Injury

A recently reported blood test for the early detection of organ transplant injury could enable more timely therapeutic intervention in transplant patients and thus help to avoid longer term damage. As described by scientists at the University Medical Center Göttingen and Chronix Biomedical, a molecular diagnostics company, the new method uses Bio-Rad Laboratories’ Droplet Digital PCR (ddPCR™) technology to overcome the obstacles of earlier tests, which were both time-consuming and costly. The method was presented at the American Association of Clinical Chemistry (AACC) 2013 annual meeting and has been accepted for publication in Clinical Chemistry.

Approximately 28,000 organ transplantations (known as grafts) are performed each year in the U.S., with another 100,000 patients on waiting lists. However, transplant patients are often subject to organ rejection: acute rejection of liver transplants within three years is nearly 22 percent, while heart and lung rejection is close to 50 percent. In addition, nearly half of all of kidney transplants fail within 10 years.

Graft-derived cell-free DNA (GcfDNA) in the circulation of transplant recipients is a potential rejection biomarker. But previous attempts to determine GcfDNA, which require parallel sequencing of donor and recipient DNA, are expensive and require a long turnaround and use of donor DNA. University Medical Center Göttingen and Chronix Biomedical researchers sought to develop a new method in an attempt to address these drawbacks.

Using ddPCR for Fast, Cost-Effective Test

The researchers applied Bio-Rad’s ddPCR technology to quantify graft-derived cfDNA in recent liver transplant patients and in stable patients who had undergone a transplant procedure more than six months earlier. ddPCR technology allowed them to develop a cost-effective and fast laboratory test that detects cfDNA being released into the blood stream by dying cells from the transplanted organ.

“GcfDNA from dying graft cells are the most direct and sensitive indicator of organ rejection and we needed an instrument that could measure it,” said Chronix Biomedical’s Chief Technology Officer and the study’s senior author, Ekkehard Schuetz, MD, PhD. “ddPCR added an additional level of reliability and precision to traditional PCR.”

Sequencing methods typically require batch sampling, but by using ddPCR, researchers are able to run single samples. Additionally, this method is reducing test time from three days or more to one day and costs by 90 percent. The study authors were able to address the need for donor DNA by preselecting SNPs that ensure enough heterogeneity between donor and recipient. The new blood test can also deliver results up to several days before the conventional aspartate aminotransferase (AST) and bilirubin tests for liver transplantation rejection, with the potential for an immediate positive impact on patient care.

“We will now be able to detect subclinical rejection and early intervention may allow us to avoid a full-blown rejection,” said Michael Oellerich, M.D., FACB, FRCPath and Lower Saxony Distinguished Professor of Clinical Chemistry at the University Medical Center Göttingen and study Principal Investigator. “This test may be useful to personalize immunosuppression and to improve long-term outcomes.”

“Detecting non-host cfDNA is the third example for the commercial potential of cfDNA diagnostics. Researchers will now be able to extend the applications from fetal cfDNA in maternal blood and personalized biomarkers for minimal residual disease in cancer to solid organ transplantation,” said Howard Urnovitz, PhD, Chronix Biomedical’s Chief Executive Officer.

“We are looking forward to the improvements in precision medicine we can offer with ddPCR and this example in transplantation highlights the diagnostic value for the technology,” said Paula Stonemetz, Director Diagnostic Business Development, Digital Biology Center, Bio-Rad Laboratories.

The researchers were awarded a National Academy of Clinical Biochemistry (NACB) Distinguished Abstract Award at the 2013 AACC annual conference. The results are part of a larger planned study to determine if cfDNA is the earliest indication of a transplant organ rejection.

Source: EurekAlert!

Fluidigm and Olink Bioscience Bring a New Level of Protein Biomarker Discovery to the Life Science Research Market

Fluidigm and Olink Bioscience have teamed up to enable interrogation of 96 samples across 92 proteins in a single run from one microliter of sample in less than a day.

The two companies will co-market the combination of their respective products — Fluidigm’s BioMarkTM HD System and Olink Bioscience’s Proseek® Multiplex technology — bringing protein research to the Fluidigm platform and Olink Bioscience’s protein detection assays to the high-throughput, high reproducibility and unparalleled sensitivity realm of Fluidigm’s real-time PCR system.

Fluidigm’s BioMark HD System and Olink Bioscience’s Proseek Multiplex technologies provide researchers with the highest throughput multiplexing solution for protein biomarker discovery in the market today. Typically, researchers are limited to working with a few tens-of-protein biomarkers at a time. Using the BioMark HD System with the Proseek Multiplex Oncology I 96×96 Kit, a researcher can simultaneously analyze 96 human samples against a panel of 92 analytes, such as growth factors, inflammatory markers, soluble receptors, or cancer antigens. With the addition of four control analytes (two incubation controls, and extension and detection controls), researchers can now obtain results for up to 9,216 reactions in just a few hours.

The first 92-plex Olink panel, available now, is focused on biomarker discovery for cancer. Panels addressing cardiovascular disease and inflammation are expected to be offered later this year.

“Protein research is so important because these biomarkers are used to monitor health states and predict treatment outcomes in patients. One of the biggest trends in the life sciences industry today is research to uncover biomarkers that can lead to companion diagnostics,” said Simon Fredriksson, Olink Bioscience president and chief executive officer. “Conventional immunoassays have been unable to scale due to increasing antibody cross-reactivity when more and more assays are run simultaneously. Olink’s Proseek Multiplex generates high quality data even in highly multiplexed formats, and using these assays in conjunction with Fluidigm’s BioMark HD System gives protein researchers easy access to unprecedented volume and quality of data.”

The Olink Bioscience assay provides a signal when pairs of antibodies equipped with DNA reporter molecules bind to their targets to create new DNA amplicons. The amplicons are subsequently quantified by high throughput real-time PCR. With PCR’s large dynamic range and superb sensitivity, in combination with Olink Bioscience’s unique protein detection assays, the opportunities are enormous for powerful analysis of multivariate biomarker patterns.

“Analyzing 92 proteins from one microliter of sample enables new biomarker discovery and validation,” said Gajus Worthington, Fluidigm president and chief executive officer. “Many sample sources, including those from biorepositories or model organisms, are limited, and researchers can simply run out before they are able to find useful biomarker panels. The combination of Fluidigm’s BioMark HD System and Olink’s Proseek Multiplex assay represents a robust new tool for the protein research community.”

Fluidigm’s BioMark HD System is a multi-application genomics and proteomics platform that provides results equivalent to the gold standards for every respective experimental approach. The system produces high-quality data from RNA, miRNA and DNA from sample sizes down to the single cell level — and now extends to proteins. The BioMark HD System performs analysis of protein expression, gene expression, copy number variation, SNP genotyping, and digital PCR.

Olink Bioscience’s Proseek Multiplex is a multivariate protein biomarker detection kit based on Olink’s proprietary Proximity Extension Assay (PEA). It uniquely allows multiplexing of immunoassays without compromising assay performance. PEA uses pairs of oligonucleotide-labeled antibodies equipped with DNA reporter molecules to bind to proteins of interest in a highly specific manner, solving the antibody cross-reactivity dilemma that plagues and limits conventional protein assays.

Source: Business Wire

Ariosa Diagnostics, CHU de Quebec and Genome Canada to Launch Large-Scale Cost-Effectiveness Study of Non-Invasive Prenatal Testing in Canada

A team of researchers led by CHU de Quebec and Universite Laval, have received CAN$10.5 million from Genome Canada, the Canadian Institutes of Health Research (CIHR), Genome Quebec and other partners to conduct a large-scale comparative effectiveness study on non-invasive prenatal screening techniques. Dr. Francois Rousseau and his team will examine current prenatal screening practices for chromosomal conditions, such as Down syndrome, among pregnant women in Canada in order to improve screening approaches and avoid unnecessary procedures.

Each year in Canada, about 450,000 women become pregnant and are offered Down syndrome prenatal screening using biochemical and ultrasound markers. The vast majority will be negative or low risk. However, for positive or high-risk results, those pregnant women are referred to amniocentesis, which is an invasive procedure done to confirm the screening result. Approximately 5% of all biochemical screening results are falsely positive, attributing to unnecessary invasive procedures that pose a 1 in 300 risk for miscarriage.

Genome Quebec is a strong supporter of public-private partnerships, such as the one developed between Ariosa Diagnostics and Dr. Francois Rousseau’s team. The PEGASUS: PErsonalized Genomics for prenatal Aneuploidy Screening USing maternal blood project, made possible through such partnerships in personalized medicine, will help meet women’s healthcare needs by giving them access to safer prenatal screening tests, explains Marc LePage, Genome Quebec’s President and CEO.

The research project aims at independently comparing the performances of different such approaches that involve various combinations of the available tools for screening of chromosomal conditions, as well as to evaluate the cost-effectiveness, the ethical and social aspects of this new technology and to identify and adapt the best implementation tools for users in the health care system. The researchers will recruit 5600 pregnant women (3600 at high-risk of trisomy conditions and 2000 at low-risk). Samples from these women will be tested in parallel using different screening approaches that involve genomic-based NIPT, but also existing or new biochemical and ultrasound screening tools. The samples will be analyzed without knowledge of the true status of the pregnancy outcome. This will provide a comprehensive evaluation of the most efficient ways to improve the prenatal screening techniques widely used today.

Ariosa Diagnostics is one of the major commercial partners for this project and will provide testing for part of this large sample that will include mainly low-risk women. The investigators have chosen the Harmony™ Prenatal Test as the commercially available NIPT assay to be included as one the various screening tests that will be compared.

Dr. Rousseau, Leader of the Project, mentions: “We are happy that Ariosa Diagnostics has joined this effort and provided a significant contribution to this Project. Ariosa’s Harmony test is the most affordable commercial NIPT available, and it was chosen by the research team for this study because it is the most likely to be used in the Canadian context.”

Dr. Thomas Musci, VP of Clinical and Medical Affairs at Ariosa remarked that: “Ariosa, as a core corporate value, is committed to furthering rigorous clinical and scientific study of cell-free DNA technology and the Harmony Prenatal Test in a variety of clinical settings and in a number of different countries. We are extremely pleased to support this collaboration with Genome Canada and fully support the goals of their research efforts.”

Dr. Serge Rivest, Director of the research Centre at the CHU de Qudbec, stated that: “The leadership of the researchers from CHU de Quebec Research Center was key in putting this exceptional academic team together to tackle one of the hottest clinical application of next-generation sequencing in the context of a population-based screening program.”

Source: Ariosa Diagnostics

Life Technologies Signs Five-Year Agreement with the FDA

Life Technologies Corporation (NASDAQ: LIFE) recently announced that it has signed a five-year agreement with the Food and Drug Administration (FDA) to accelerate and advance food safety testing of E. coli and Salmonella, two foodborne contaminants commonly associated with outbreaks and/or recalls.

The collaboration consists of three distinctive projects:

  • Life Technologies will design and validate new food safety tests for the detection and identification of foodborne pathogens, with pathogen strains provided by the FDA.
  • Life Technologies will design and validate a complete workflow for food pathogen detection on the Ion PGM™ platform, while optimizing sample preparation methods.
  • The FDA will have the opportunity to evaluate new disruptive technology platforms by Life Technologies for the use in pathogen detection.

Life Technologies will use its bioinformatics resources to independently develop real-time PCR (polymerase chain reaction) assays against unique E. coli and Salmonella targets in collaboration with the FDA. It will then provide the test results for these targets to the FDA for further validation.

The FDA will use Life Technologies’ Ion PGM™ Sequencer to generate whole genome sequence information from defined bacteria, and for strains that will be excluded from detection. Life Technologies’ bioinformatics team will then analyze the genomic information and provide assays or tests to the FDA for further validation and analysis. Whole genome sequences generated under the collaboration will be added to the National Institutes of Health Genbank so they can be accessed by the food safety research community.

“We are excited to be entering this cooperative research and development agreement with the FDA as we have been working alongside them in one capacity or the other for over 10 years,” said Nir Nimrodi, Vice President and General Manager for Food Safety and Animal Health at Life Technologies. “The FDA will call on us, particularly when it comes to developing rapid detection kits. This agreement allows them to have new rapid track and trace products for rapid identification of foodborne contaminants.”

Lastly, the FDA will validate and test Life Technologies’ next-generation sequencing technologies for Salmonella investigations as part of its effort to develop new rapid detection tools that can improve the public health response to future outbreaks.

Life Technologies has a strong history of combating pathogen outbreaks. In 2011, Life Technologies developed a custom assay to accurately detect the highly pathogenic E. coli 0104:H4 bacterium that spread through Europe, killing hundreds of people.

“The assay was developed using samples supplied by scientists at University Hospital Munster, who completed the DNA sequencing and analysis work on the deadly E. coli strain using the Ion PGM™ Sequencer,” said Maneesh Jain, Ion Torrent Vice President of Marketing for Life Technologies. “Now the FDA will validate and test this same technology to understand the molecular relationship within the Salmonella pathogen in the hopes to prevent future outbreaks from occurring.”

The PGM™ offers scientists the simplest and fastest workflow, lowest cost-per-sample, and highest sensitivity for multiple basic and clinical research applications.

Source: Life Technologies

Potential Clue Associated with Aggressive Prostate Cancer Identified by Rutgers Investigators

Prostate cancer is one of the most common forms of cancer in men and the leading cause of cancer deaths in white, African-American and Hispanic men, according to the Centers for Disease Control. Current treatment of prostate cancer targets androgens, hormones which promote the growth and spread of cancer cells. However, it remains unclear why, despite treatment, some prostate cancers progress and may become fatal. Researchers at Robert Wood Johnson Medical School, part of Rutgers, The State University of New Jersey, who are studying the underlying mechanisms that cause invasive tumor growth have identified a key transcription factor, a protein which regulates the flow of information from DNA, that is over-produced in treatment-resistant prostate cancer, as well as the two protein kinases that trigger the process. This finding, published and highlighted on the cover of the July issue of Molecular Cancer Research, a journal of the American Association for Cancer Research (AACR) could be utilized to develop treatments for prostate cancer that is resistant to current therapies.

The research team, led by Joseph Fondell, PhD, associate professor of pharmacology, found that in clinically localized human prostate cancer — cancer that is confined to the prostate and pelvic area — the key transcription factor termed MED1 is overexpressed, meaning that significantly more MED1 is produced than is typical. According to Fondell, the finding potentially could be used as a biomarker in cancer screenings, indicating to oncologists that the prostate cancer has become aggressive. “As MED1 is a known co-activator of androgen receptors, the overexpression of MED1 is thought to facilitate alternative gene expression patterns that drive treatment-resistant cancer cell growth in the prostate,” Fondell said.

“Our study showed for the first time that MED1 expression is elevated in malignant cells of a statistically significant number of patients with clinical prostate cancer and that this overexpression correlates with an increase in cancer cell growth and invasiveness,” said Feng Jin, PhD, a former graduate student in Fondell’s lab and first author on the study. “In addition to accelerated tumor growth, our study showed that overexpression of MED1 may also be involved with inflammation of the prostate.”

Further study of the process using mouse models that mimic human prostate cancer, showed that two protein kinases, ERK and PI3K/AKT, were overactive and responsible for MED1 overproduction, ultimately accelerating the progression and spread of prostate cancer.

“Whereas the current treatment approach for prostate cancer is to prohibit androgen production and signaling, our findings indicate that MED1 could represent a novel target for new therapies that stop the process at the molecular level, before prostate cancer can progress to an advanced stage,” added Fondell, who also is a member of Rutgers Cancer Institute of New Jersey.

Study: ERK and AKT Signaling Drive MED1 Overexpression in Prostate Cancer in Association with Elevated Proliferation and Tumorigenicity [Molecular Cancer Research]

Source: Robert Wood Johnson Medical School