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Posts Tagged ‘Roche Diagnostics’


Roche is developing a high-throughput low cost sequencer for NGS

Reporter: Stephen J. Williams, PhD

 

Reported from Diagnostic World News

Long-Read Sequencing in the Age of Genomic Medicine

 

 

By Aaron Krol

December 16, 2015 | This September, Pacific Biosciences announced the creation of the Sequel, a DNA sequencer half the cost and seven times as powerful as its previous RS II instrument. PacBio, with its unique long-read sequencing technology, had already secured a place in high-end research labs, producing finished, highly accurate genomes and helping to explore the genetic “dark matter” that other next-generation sequencing (NGS) instruments miss. Now, in partnership with Roche Diagnostics, PacBio is repositioning itself as a company that can serve hospitals as well.

“Pseudogenes, large structural variants, validation, repeat disorders, polymorphic regions of the genome―all those are categories where you practically need PacBio,” says Bobby Sebra, Director of Technology Development at the Icahn School of Medicine at Mount Sinai. “Those are gaps in the system right now for short-read NGS.”

Mount Sinai’s genetic testing lab owns three RS II sequencers, running almost around the clock, and was the first lab to announce it had bought a Sequel just weeks after the new instruments were launched. (It arrived earlier this month and has been successfully tested.) Sebra’s group uses these sequencers to read parts of the genome that, thanks to their structural complexity, can only be assembled from long, continuous DNA reads.

There are a surprising number of these blind spots in the human genome. “HLA is a huge one,” Sebra says, referring to a highly variable region of the genome involved in the immune system. “It impacts everything from immune response, to pharmacogenomics, to transplant medicine. It’s a pretty important and really hard-to-genotype locus.”

Nonetheless, few clinical organizations are studying PacBio or other long-read technologies. PacBio’s instruments, even the Sequel, come with a relatively high price tag, and research on their value in treating patients is still tentative. Mount Sinai’s confidence in the technology is surely at least partly due to the influence of Sebra―an employee of PacBio for five years before coming to New York―and Genetics Department Chair Eric Schadt, at one time PacBio’s Chief Scientific Officer.

Even here, the sequencers typically can’t be used to help treat patients, as the instruments are sold for research use only. Mount Sinai is still working on a limited number of tests to submit as diagnostics to New York State regulators.

Physician Use

Roche Diagnostics, which invested $75 million in the development of the Sequel, wants to change that. The company is planning to release its own, modified version of the instrument in the second half of 2016, specifically for diagnostic use. Roche will initially promote the device for clinical studies, and eventually seek FDA clearance to sell it for routine diagnosis of patients.

In an email to Diagnostics World, Paul Schaffer, Lifecycle Leader for Roche’s sequencing platforms division, wrote that the new device will feature an integrated software pipeline to interpret test results, in support of assays that Roche will design and validate for clinical indications. The instrument will also have at least minor hardware modifications, like near field communication designed to track Roche-branded reagents used during sequencing.

This new version of the Sequel will probably not be the first instrument clinical labs turn to when they decide to start running NGS. Short-read sequencers are sure to outcompete the Roche machine on price, and can offer a pretty useful range of assays, from co-diagnostics in cancer to carrier testing for rare genetic diseases. But Roche can clear away some of the biggest barriers to entry for hospitals that want to pursue long-read sequencing.

Today, institutions like Mount Sinai that use PacBio typically have to write a lot of their own software to interpret the data that comes off the machines. Off-the-shelf analysis, with readable diagnostic reports for doctors, will make it easier for hospitals with less research focus to get on board. To this end, Roche acquired Bina, an NGS analysis company that handles structural variants and other PacBio specialties, in late 2014.

The next question will be whether Roche can design a suite of tests that clinical labs will want to run. Long-read sequencing is beloved by researchers because it can capture nearly complete genomes, finding the correct order and orientation of DNA reads. “The long-read technologies like PacBio’s are going to be, in the future, the showcase that ties it all together,” Sebra says. “You need those long reads as scaffolds to bring it together.”

But that envisions a future in which doctors will want to sequence their patients’ entire genomes. When it comes to specific medical tests, targeting just a small part of the genome connected to disease, Roche will have to content itself with some niche applications where PacBio stands out.

Early Applications

“At this time we are not releasing details regarding the specific assays under development,” Schaffer told Diagnostics World in his email. “However, virology and genetics are a key focus, as they align with other high-priority Roche Diagnostics products.”

Genetic disease is the obvious place to go with any sequencing technology. Rare hereditary disorders are much easier to understand on a genetic level than conditions like diabetes or heart disease; typically, the pathology can be traced back to a single mutation, making it easy to interpret test results.

Some of these mutations are simply intractable for short-read sequencers. A whole class of diseases, the PolyQ disorders and other repeat disorders, develop when a patient has too many copies of a single, repetitive sequence in a gene region. The gene Huntingtin, for example, contains a long stretch of the DNA code CAG; people born with 40 or more CAG repeats in a row will develop Huntington’s disease as they reach early adulthood.

These disorders would be a prime target for Roche’s sequencer. The Sequel’s long reads, spanning thousands of DNA letters at a stretch, can capture the entire repeat region of Huntingtin at a stretch, unlike short-read sequencers that would tend to produce a garbled mess of CAG reads impossible to count or put in order.

Nonetheless, the length of reads is not the only obstacle to understanding these very obstinate diseases. “The entire category of PolyQ disorders, and Fragile X and Huntington’s, is really important,” says Sebra. “But to be frank, they’re the most challenging even with PacBio.” He suggests that, even without venturing into the darkest realms of the genome, a long-read sequencer might actually be useful for diagnosing many of the same genetic diseases routinely covered by other instruments.

That’s because, even when the gene region involved in a disease is well known, there’s rarely only one way for it to go awry. “An example of that is Gaucher’s disease, in a gene called GBA,” Sebra says. “In that gene, there are hundreds of known mutations, some of which you can absolutely genotype using short reads. But others, you would need to phase the entire block to really understand.” Long-read sequencing, which is better at distinguishing maternal from paternal DNA and highlighting complex rearrangements within a gene, can offer a more thorough look at diseases with many genetic permutations, especially when tracking inheritance through a family.

“You can think of long-read sequencing as a really nice way to supplement some of the inherited panels or carrier screening panels,” Sebra says. “You can also use PacBio to verify variants that are called with short-read sequencing.”

Virology is, perhaps, a more surprising focus for Roche. Diagnosing a viral (or bacterial, or fungal) infection with NGS only requires finding a DNA read unique to a particular species or strain, something short-read sequencers are perfectly capable of.

But Mount Sinai, which has used PacBio in pathogen surveillance projects, has seen advantages to getting the full, completely assembled genomes of the organisms it’s tracking. With bacteria, for instance, key genes that confer resistance to antibiotics might be found either in the native genome, or inside plasmids, small packets of DNA that different species of bacteria freely pass between each other. If your sequencer can assemble these plasmids in one piece, it’s easier to tell when there’s a risk of antibiotic resistance spreading through the hospital, jumping from one infectious species to another.

Viruses don’t share their genetic material so freely, but a similar logic can still apply to viral infections, even in a single person. “A virus is really a mixture of different quasi-species,” says Sebra, so a patient with HIV or influenza likely has a whole constellation of subtly different viruses circulating in their body. A test that assembles whole viral genomes—which, given their tiny size, PacBio can often do in a single read—could give physicians a more comprehensive view of what they’re dealing with, and highlight any quasi-species that affect the course of treatment or how the virus is likely to spread.

The Broader View

These applications are well suited to the diagnostic instrument Roche is building. A test panel for rare genetic diseases can offer clear-cut answers, pointing physicians to any specific variants linked to a disorder, and offering follow-up information on the evidence that backs up that call.

That kind of report fits well into the workflows of smaller hospital labs, and is relatively painless to submit to the FDA for approval. It doesn’t require geneticists to puzzle over ambiguous results. As Schaffer says of his company’s overall NGS efforts, “In the past two years, Roche has been actively engaged in more than 25 partnerships, collaborations and acquisitions with the goal of enabling us to achieve our vision of sample in to results out.”

But some of the biggest ways medicine could benefit from long-read sequencing will continue to require the personal touch of labs like Mount Sinai’s.

Take cancer, for example, a field in which complex gene fusions and genetic rearrangements have been studied for decades. Tumors contain multitudes of cells with unique patchworks of mutations, and while long-read sequencing can pick up structural variants that may play a role in prognosis and treatment, many of these variants are rarely seen, little documented, and hard to boil down into a physician-friendly answer.

An ideal way to unravel a unique cancer case would be to sequence the RNA molecules produced in the tumor, creating an atlas of the “transcriptome” that shows which genes are hyperactive, which are being silenced, and which have been fused together. “When you run something like IsoSeq on PacBio and you can see truly the whole transcriptome, you’re going to figure out all possible fusions, all possible splicing events, and the true atlas of reads,” says Sebra. “Cancer is so diverse that it’s important to do that on an individual level.”

Occasionally, looking at the whole transcriptome, and seeing how a mutation in one gene affects an entire network of related genes, can reveal an unexpected treatment option―repurposing a drug usually reserved for other cancer types. But that takes a level of attention and expertise that is hard to condense into a mass-market assay.

And, Sebra suggests, there’s another reason for medical centers not to lean too heavily on off-the-shelf tests from vendors like Roche.

Devoted as he is to his onetime employer, Sebra is also a fan of other technologies now emerging to capture some of the same long-range, structural information on the genome. “You’ve now got 10X Genomics, BioNano, and Oxford Nanopore,” he says. “Often, any two or even three of those technologies, when you merge them together, can get you a much more comprehensive story, sometimes faster and sometimes cheaper.” At Mount Sinai, for example, combining BioNano and PacBio data has produced a whole human genome much more comprehensive than either platform can achieve on its own.

The same is almost certainly true of complex cases like cancer. Yet, while companies like Roche might succeed in bringing NGS diagnostics to a much larger number of patients, they have few incentives to make their assays work with competing technologies the way a research-heavy institute like Mount Sinai does.

“It actually drives the commercialization of software packages against the ability to integrate the data,” Sebra says.

Still, he’s hopeful that the Sequel can lead the industry to pay more attention to long-read sequencing in the clinic. “The RS II does a great job of long-read sequencing, but the throughput for the Sequel is so much higher that you can start to achieve large genomes faster,” he says. “It makes it more accessible for people who don’t own the RS II to get going.” And while the need for highly specialized genetics labs won’t be falling off anytime soon, most patients don’t have the luxury of being treated in a hospital with the resources of Mount Sinai. NGS companies increasingly see physicians as some of their most important customers, and as our doctors start checking into the health of our genomes, it would be a shame if ubiquitous short-read sequencing left them with blind spots.

Source: http://diagnosticsworldnews.com/2015/12/16/long-read-sequencing-age-genomic-medicine.aspx

 

 

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Reporter: Aviva Lev-Ari, PhD, RN

Updated 3/10/2013

Since August 25, 2012, when the ESC: New Definition of MI Unveiled was reviewed by Robert Jasmer, MD; Associate Clinical Professor of Medicine, University of California, San Francisco as was reported  By Chris Kaiser, Cardiology Editor, MedPage Today,  a new discussion emerged by ACC asking if FFR CT is Ready for prime time or not?

By Lisa Fratt
Mar 09, 2013

SAN FRANCISCO—Is there a better way to measure fractional flow reserve (FFR), Bon-Kwon Koo, MD, of Seoul National University queried a crowded room March 9 during an educational session at the American College of Cardiology (ACC) scientific session.

The current model is good for patients, safe and effective, Koo said. However, it requires an invasive procedure and is expensive. FFR CT may provide a method to measure FFR without an invasive procedure.

FFRCT extracts geometry from a CT scan to determine boundary conditions and fluid properties. In addition, velocity and pressure can be calculated. The hitch is that a supercomputer is required to solve the blood flow equation, said Koo. The results provide anatomical and functional data, thus giving a possible answer to the question at hand.

FFRCT may change daily practice in several ways. Most importantly, it may be a novel, fast, risk-free, noninvasive cost-saving way to measure FFR and identify patients who may not need to be sent to the cath lab for stenting or PCI. It can provide information to help surgeons plan strategies before invasive procedures, bypass procedures or interventional procedures. Noninvasive CT-derived FFR also can predict the functional significance of coronary lesions.

Despite its promise, however, FFR CT is not ready for prime time, Koo said. FFR CT depends on the diagnostic accuracy of coronary CT angiography stenosis, which is less than true stenosis. With current technologies, true stenosis provides the required diagnostic accuracy.

FFRCT is promising, but further development of the technology is required, Koo concluded.

http://www.cardiovascularbusiness.com/topics/imaging/acc-ffrct—ready-prime-time-or-not

ESC: New Definition of MI Unveiled

By Chris Kaiser, Cardiology Editor, MedPage Today

Published: August 25, 2012

Reviewed by Robert Jasmer, MD; Associate Clinical Professor of Medicine, University of California, San Francisco

MUNICH — An international, multispecialty task force has published a new definition of myocardial infarction that was prompted by the new generation of highly sensitive cardiac troponin (cTn) assays.

The highly sensitive assays are capable of detecting cTn in conditions other than MI, such as pulmonary embolism, cardiomyopathy, and left bundle branch block, and so result in false positives, according to the task force writing group.

The expert consensus document dips into a controversial area by setting levels of cTn for MI associated with percutaneous coronary intervention (PCI) and coronary artery bypass grafting (CABG).

“This is one of the most controversial areas in the definition of myocardial infarction,” Anthony DeMaria, MD, from the University of California in San Diego and editor-in-chief of the Journal of the American College of Cardiology, told MedPage Today.

“There are a large number of people undergoing PCI in the setting of an acute MI. It’s almost impossible to know whether a subsequent increase in troponin was part and parcel of the acute MI or related to the procedure itself,” DeMaria said.

The consensus document, titled “Third Universal Definition of Myocardial Infarction,” set the cTn levels for MI associated with PCI as elevation of troponin greater than 5 times the 99th percentile upper reference limit (URL) in patients with normal baseline levels or a rise in troponin values greater than 20% if the baseline values are elevated and are stable or falling.

“Some people speculate that troponin may be too sensitive in this situation and what is needed is evidence that an elevation of some degree of troponin following a procedure actually results in some alteration of the natural history of the patient,” DeMaria said. “In other words, the definition of acute MI after a procedure really is of significance if it increases the risk of subsequent events such as death.”

In CABG, the task force set the troponin values as greater than 10 x 99th percentile URL during the first 48 hours when baseline values are normal.

DeMaria said there are several ongoing studies examining the correlation of elevated cTn with subsequent events. As this is the third definition of MI since 2000, there most likely will be more refinements as new data emerge, he said.

The document is being copublished online in several journals including the Journal of the American College of CardiologyCirculation, the European Heart Journal, and Global Heart.

The task force was in touch with the FDA during the development of this new definition, which means it could be used as the basis for clinical trial protocols designed according to FDA regulations.

“A universal definition for MI is of great benefit for clinical studies, since it will allow a standardized approach for interpretation and comparison across different trials,” the task force writing group explained.

When different definitions have been used in trials, it hampers “comparison and generalization between these trials,” they said.

Also of significance in this document is the inclusion of imaging as a means to identify or confirm an MI. The document spells out the strengths of echocardiography, nuclear imaging, MRI, and CT in the setting of acute MI.

“Imaging is playing an increasingly important role,” DeMaria said. “In the absence of focal symptoms or with an inconclusive ECG, it’s important to recognize the concomitant potential of ancillary measures, primarily imaging, to help with the diagnosis of a myocardial infarction.”

Thygesen reported relationships with Edwards Lifesciences, Servier, St. Jude Medical, Roche Pharma, and Roche Diagnostics. Her co-authors and reviewers reported relationships with Bayer Healthcare, Daiichi Sankyo, Johnson & Johnson, sanofi aventis, Servier, Novartis, Boehringer-Ingelheim, Genzyme, Eli Lilly, OrthoClinical Diagnostics, Abbott Laboratories, Alere, Brahms, Siemens Healthcare, Roche Pharma, Radiometer, BioRad, Diagenics, Response Medical, Takeda Pharmaceuticals, Regado Biosciences, Bristol-Myers Squibb, Merck Sharp and Dohme, GlaxoSmithKline, Merck, Portola Pharmaceuticals, AstraZeneca, Regado Biosciences, Scios, Ortho-Biotech, Pfizer, Kai Pharmaceuticals, Iroko Cardio, Philips, GE Healthcare, Boston Scientific, Lantheus, Medtronic, St. Jude Medical, Biotronik, Impulse Dynamics, Edwards Lifesciences, Health System Networks, Health Station Networks, Insight Telehealth Systems, Elsevier Sciences, Gilead, Evolva, Medicines Company, F. Hoffman La Roche, Torrent, Vifor International, Corthera, Nanosphere, Bayer Schering Pharma, Cardiorentis, Molecular Insight Pharmaceuticals, Berlin Chemie, Menarini, Cordis, Beckman Coulter, Amgen, Critical Diagnostics, Tethys Bioscience, Roche Diagnostics, bioMérieux, Genentech, Ikaria, Singulex, BG Medicine, Shionogi, Amylin, DiaDexus, Orion, WebMD, theheart.org, Pozen, Maquet, BHFZ, Covidien, Rapidscan, Actelion, Athera, Symetis, Schering-Plough, OrbusNeich, Terumo, Cardio3 Biosciences, Micell, Ablynx, Therabel, Kowa, Zentiva, Chugai Pharma, Automedics Medical Systems, Essentialis, Biosensors, Vascular Solutions, Zoll Medical, JaBA Recordati, Actavis, PharmaSwiss, Eisai, Medscape, Accumetrics, Bial Portela, AGA, Novo-Nordisk, Janssen-Cilag, Valtech, Otsuka Pharmaceuticals, Meda Pharma, CEPHALON, Intracellular Therapies USA, Santhera, TROPHOS, Pierre-Fabre, and Lundbeck.

DeMaria reported relationships with Gilead, ResMed Foundation, Lantheus, Cardiovascular Biotherapeutics, Angioblast Systems, General Electric Medical Systems, and Cardionet.

Primary source: European Heart Journal

Source reference:
Thygesen K, et al “Third universal definition of myocardial infarction” Eur Heart J 2012; DOI: 10.1093/eurheartj/ehs184.

ESC: FFR CT Has Potential for Tagging Ischemia

By Chris Kaiser, Cardiology Editor, MedPage Today

Published: August 26, 2012

Reviewed by Robert Jasmer, MD; Associate Clinical Professor of Medicine, University of California, San Francisco

MUNICH — Using CT imaging to assess the hemodynamic significance of coronary lesions is “promising” but needs more research before it displaces conventional invasive fractional flow reserve (FFR), researchers said.

Using FFR as the reference standard, FFRCTplus CT angiography (CTA) had good sensitivity (90%) and negative predictive value (84%) on a per patient basis for detecting ischemia, which indicates a low rate of false-negative studies, according to James K. Min, MD, of Cedars-Sinai Heart Institute in Los Angeles, and colleagues.

Although FFRCT plus CTA were superior to CTA alone, the specificity (54%) and negative predictive value (67%) of the combination remained low compared with conventional FFR, indicating that a considerable number of false-positive studies would endure, Min reported here during a Hot-Line session at the European Society of Cardiology meeting.

The results of this proof of concept study show that FFRCT can “impart considerable discriminatory power” to detect and exclude ischemia in patients with suspected CAD, Min said.

However, future studies should be conducted to determine the cost-effectiveness of FFRCT in guiding decisions to stent, particularly given the potentially high false-positive rate, he added.

“Non-invasive FFR is a dream for all interventional cardiologists,” said study discussant Jean-Pierre Bassand, MD, of the University Hospital Jean-Minjoz in Besançon, France. Although Bassand praised the DeFACTO study, he expressed concern about the discrepancy between the accuracy of FFR versus FFRCT.

For example, compared with FFR, the sensitivity and specificity of FFRCT in cases of greater than 90% or less than 30% stenosis were 83% and 76%, respectively. The per-vessel correlation of FFRCT to FFR was 0.63.

“What matters is the correlation with FFR,” he concluded.

A single non-invasive imaging test that can identify obstructive coronary artery disease (CAD) and determine the physiological significance of those lesions would be ideal. At present, nuclear stress imaging fulfills the first part, but it cannot label stenoses as hemodynamically significant or not. Also, nuclear stress testing suffers from high rates of both false-negative and false-positive studies, Min said.

The results of this study are in line with stress imaging: per patient diagnostic accuracy of 73% (95% CI 67% to 78%). Min said that studies are being designed to compare FFRCT plus CTA with stress imaging.

“For patients considered for invasive therapy, this type of test could help exclude those who don’t need to be stented,” Spencer King III, MD, of St. Joseph’s Hospital in Atlanta told MedPage Today.

“The excitement about this CT approach is that it moves things closer to being able to assess physiology and anatomy in a single non-invasive test,” added King, who is also a past president of the American College of Cardiology.

However, the process of calculating the FFR values from CT data currently takes about 6 hours, Min told MedPage Today. The CT data are sent offsite to HeartFlow, the company that makes the software. Whether such processing would be done onsite in the future is not yet determined, Min said. He also expects the processing time to drop to about 2 hours by the year’s end.

HeartFlow has already received EU mark to use the software in Europe and is in the process of applying for FDA approval, Min said.

Conventional FFR uses a pressure wire inserted through the groin to the coronary arteries to determine the hemodynamic significance of lesions. The same data can be gleaned during a typical CTA exam with software that calculates computational fluid dynamics,without additional radiation exposure. The median radiation exposure among the study centers was 6.4 mSv (range 4.4 to 15 mSv).

The original FAME study found the use of FFR to guide stenting was better than relying on angiography alone in patients with multivessel disease. A second study, FAME II, was stopped early because of the overwhelming benefit seen in patients with stable CAD when FFR guided stenting versus patients randomized to optimal medical therapy.

Because FFRCT is a novel technique, it has not been adequately evaluated in its ability to identify patients with ischemia, Min said.

The researchers therefore designed the DeFACTO (Determination of Fractional Flow Reserve by Anatomic Computed Tomographic Angiography) study, which sought to evaluate the accuracy of FFRCT while using invasive FFR as the reference standard.

The study was also simultaneously published online in the Journal of the American Medical Association.

The 252 patients with suspected or known CAD were recruited from 17 centers in five countries between October 2010 and October 2011. They were scheduled to undergo diagnostic catheter angiography.

The mean age of patients was 63, 70% were men, and a majority were white. Nearly half of the patients had obstructive CAD (>50% stenosis).

Among 615 study vessels, 271 had less than 30% stenosis and 101 had at least 90% stenosis. Invasive coronary angiography and FFR identified 46.5% of 408 vessels with obstructive CAD, while CT and FFRCT identified 52.3% of 406 vessels.

A total of 172 patients had an FFR value <0.80, which indicates an ischemic lesion.

The diagnostic accuracy of FFRCT plus CT was 73% (95% CI 67% to 78%), but this did not meet the prespecified primary endpoint of greater than 70% of the lower bound of the 95% confidence interval, Min said.

However, Min emphasized that FFRCT was superior to CTA alone in all categories.

The researchers concluded that the results show the potential of FFRCT as a “promising” non-invasive tool to identify ischemia.

King added that despite not meeting the prespecified primary endpoint, “it’s an encouraging early study.”

This study was funded by HeartFlow

Min reported relationships with GE Healthcare and Philips Medical. Some of his co-authors reported relationships with GE Healthcare, Siemens Medical Systems, Lantheus Medical Imaging, Boston Scientific, Merck, Abbott Vascular, Medtronic, Cordis, Eli Lilly, Daiichi Sankyo, Bristol-Myers Squibb, and sanofi-aventis.

King reporeted relationships with Merck & Company, Wyeth Pharmaceuticals, Celonova Biosciences, and Northpoint Domain.

 

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