Nucleic Acid-Based Circulating Biomarkers for Cancer Diagnostics Become Reality

  • Circulating Tumor Cell Blood Tests Approved by FDA
  • Circulating DNA Stool Test Approved for Colorectal Screening to Avoid Colonoscopy
  • Circulating mRNA Urine Test Approved for use to Reduce the Total Number of Unnecessary Prostate Biopsies

Backstory

Taken from sysmex-inostics.com 

Taken from sysmex-inostics.com

According to the NIH National Cancer Institute website, ~1.6 million persons in the U.S. alone will be diagnosed with cancer this year. A very important key to survival is early detection. To enable significantly earlier diagnosis compared to manifestation of clinical symptoms, researchers have been focusing on finding DNA or RNA biomarkers that are circulating in blood, which is readily available and relatively noninvasive compared to traditional biopsies.

exosomesSome of the basic processes underlying this paradigm-shift in cancer diagnostics are depicted in the simplified cartoon wherein tumor cells, or components thereof, pass into the bloodstream. This leads to circulating tumor cells (CTCs) and cell-free circulating tumor DNA (ctDNA) to investigate and differentiate from their normal counterparts as sources of potential biomarkers.

That task is much easier said than done because of the need to sort through all of the normal components in blood, as well as deal with circulating cells and DNA derived from apoptosis (aka programed cell death) and necrosis that are normal ongoing “background” to contend with. In addition to CTCs and ctDNA, there is active cellular excretion of small (30-100 nm) exosome particles as depicted in the following graphic. Consequently, gene-encoding mRNAs, gene-regulating micro RNAs (miRNA), and potentially other exosomal components, can serve as diagnostic biomarkers.

Snapshots of Recent Commercial Diagnostic Products

My search of PubMed for publications indexed to “circulating biomarkers” AND “cancer” led to ~9,000 items, the vast majority of which have appeared during the past decade at an accelerating annual rate.  In fact, there were ~1,000 publications in 2014 alone—that’s roughly 3 such publications every day! Those interested in perusing this mountain of information later can use this link, as my intention here is to comment on resultant commercial diagnostic products, each of which provides all-important early diagnosis using a simple blood test, or urine or stool.

CTCs

In one of my blogs last year, I asserted that liquid biopsies were (metaphorically) clinically valuable “liquid gold” in a modern day Gold Rush. My evidence for the “rush” was a then recent review in Clinical Chemistry stating that “the detection and molecular characterization of CTCs are one of the most active areas of translational cancer research, with >400 clinical studies having included CTCs as a biomarker.” In that vein—double pun intended—who’s struck it rich, so to speak, commercially?

Taken from journal.frontiresin.org

Taken from journal.frontiresin.org

The answer is Veridex, which developed the CELLSEARCH® CTC Test that has the added distinction of being the first FDA-approved in vitro diagnostic (IVD) test for capturing and counting CTCs to determine the prognosis of patients (in this case for metastatic breast, colorectal or prostate cancer). This test utilizes magnetic capture of cancer-specific antibodies as depicted below.  Veridex was subsequently acquired by Jansen Diagnostics, which now offers a complete system for CELLSEARCH® CTC Test comprising sample collection, sample preparation, and sample analysis using unique immuno-magnetic and fluorescence imaging technology.

In addition, a Swiss molecular diagnostics company, Novigenix, offers its blood tests for early detection of cancer. Colox®, its lead product, is designed to significantly reduce mortality from colorectal cancer through early detection and follow-up colonoscopy. Novigenix’s technology is based on predictive gene expression profiles of circulating blood cells and tumor-derived protein markers.

Taken from Soper and coworkers in Chem. Commun. (2015).

Taken from Soper and coworkers in Chem. Commun. (2015).

Although not yet a diagnostic device, Prof. Steven Soper at UNC-Chapel Hill and a team of coworkers have recently published methods whereby captured CTCs can be enzymatically released for further analysis. This release procedure (depicted right) features use of an oligonucleotide linker containing uracil (U) that is cleaved by USER™, which consists of a mixture of uracil DNA glycosylase and DNA glycosylase-lyase endonuclease VIII.

ctDNA Biomarkers for Colon Cancer Screening

That ctDNA can provide promising biomarkers for noninvasive assessment of cancer has been successfully translated into a commercial product by Trovagene, which tests for ctDNA in urine or blood, and claims to have been the first company to have recognized the diagnostic value of ctDNA.

In addition, Cologuard® (developed by Exact Sciences in Madison, WI) was approved by the FDA as the first stool-based colorectal screening test that detects red blood cells and DNA mutations that may indicate colon cancer or precursors to cancer. Its commercials are frequently seen on TV. Given the inconvenient colon-cleansing required of patients prior to the also unpleasant invasiveness of colonoscopy, it’s not surprising that more and more persons are opting to use this new test.

In fact, Exact Sciences recently reported that during the first quarter of 2016, the company completed approximately 40,000 Cologuard® tests, an increase of more than 260% compared to approximately 11,000 tests completed in the same quarter of 2015. The cumulative number of physicians ordering Cologuard® since launch expanded to more than 32,000. Finding a doctor is relatively easy, as I found out when I located a gastrointestinal (GI) specialist near me who was also in my network—yeh!

Given the high incidence rate of colon cancer, and the traditionally recommended screening process, it was necessary for Exact Sciences to obtain compelling data in a large clinical study. An FDA announcement stated that the safety and effectiveness of Cologuard® was established in a clinical trial that screened 10,023 subjects. The trial compared the performance of Cologuard® to the fecal immunochemical test (FIT), a commonly used non-invasive screening test that detects blood in the stool. Cologuard® accurately detected cancers and advanced adenomas more often than the FIT test.

Other ctDNA Biomarkers

PlasmaSelect-R™ offered by Personal Genomics Diagnostics, which is a service company founded by experts at Johns Hopkins University, analyzes ctDNA in blood for genetic alterations in cancer based on a targeted panel of 63 well-characterized cancer genes. Cell-free DNA is extracted from plasma using proprietary methods for low-abundance sample DNA, and processed using a proprietary capture process for high-coverage next-generation sequencing to allow tumor specific mutations, amplifications, and translocations to be identified with a high sensitivity (allele fractions as low as 0.10%) and specificity. The company states that its “services further the understanding of cancer and facilitate the development of new diagnostics and therapeutics through our pioneering research approaches and novel technologies.” 

In June 2016, Roche announced that the FDA approved the cobas® EGFR Mutation Test v2 for use with plasma samples, as a companion diagnostic for the non-small cell lung cancer (NSCLC) therapy, Tarceva®. It’s important to recognize that this is the first FDA approval of a liquid biopsy test as an aid in clinical decisions, and makes it the only companion diagnostic that is FDA-approved for the detection of the epidermal growth factor receptor (EGFR) gene in tumor DNA derived from plasma (or tumor tissue). NSCLC patients who have EGFR exon 19 deletions or L858R mutations are candidates for the EGFR-targeted therapy Tarceva® (erlotinib) in first-line treatment.

Circulating RNA and miRNA

The discoveries in 1999-2000 of tumor-derived RNA in the blood of cancer patients sparked a new field for studying gene expression noninvasively using quantitative reverse transcription-PCR (qRT-PCR) and then next-generation sequencing. The existence of circulating RNA was surprising because ribonucleases are present in blood. However, mechanisms that protect circulating RNA reportedly include complexation to lipids, proteins, lipoproteins, or nucleosomes, and protection within apoptotic bodies or other vesicular structures.

Cleverly named Molecular Stethoscope is a newish startup co-founded by uber-famous Drs. Stephen Quake and Eric Topol. The company has leveraged Quake’s finding that genome-wide analysis of circulating RNA shows tissue-specific signatures from all of the major organs can be monitored in blood, and Topol’s finding that such signatures can be used to predict imminent occurrence of a heart attack. Coronary artery disease, neurodegenerative diseases, and autoimmune/inflammatory diseases are the company’s current objectives. I’m guessing, however, that cancer might be added or licensed.

My search of the literature indicates that there are far more publications on circulating miRNA, presumably due to its greater abundance resulting from its small size and/or binding to miRNA-related proteins. The biogenesis of miRNA is depicted below.

Taken from nature.com

Taken from nature.com

A review and prospectus for circulating miRNA applied to cancer has been recently published by Bertoli et al. in an article entitled MicroRNAs: New Biomarkers for Diagnosis, Prognosis, Therapy Prediction and Therapeutic Tools for Breast Cancer. From my search of this emerging field, some exemplary commercial endeavors are as follows.

The first blood-based cancer diagnostic to exploit exosomes became commercially available in the U.S. in January 2016 via launch of ExoDx Prostate(IntelliScore) by Cambridge, MA-based Exosome Diagnostics. As reported by a large team of medical experts in JAMA Oncology, qRT-PCR was used to compare the urine exosome 3-gene expression with biopsy outcomes in patients with a range of low-to-high prostate-specific antigen (PSA) levels (2 to20 ng/mL).

Taken from nature.com

Taken from nature.com

The investigators concluded that this qRT-PCR assay using urine was associated with improved identification of patients with higher-grade prostate cancer among men with elevated PSA levels and could reduce the total number of unnecessary biopsies from the ~1M total annual biopsies. The complications that have been associated with unnecessary biopsy and overtreatment range from erectile dysfunction and incontinence, to infections, sepsis and serious cardiovascular events.

At the other end of the commercial spectrum, so to speak, startup Miroculus aims to aid in the early diagnosis of cancer by making a low-cost, open-source, decentralized diagnostic they called Miriam pictured below. Their goal is for untrained workers in clinics around the world to be able to use Miriam to screen for cancer.

Taken from miroculus.com

Taken from miroculus.com

Miriam made its—or more gender specific—her public debut at the TEDGlobal conference in Rio De Janeiro in 2014 with TED curator Chris Anderson calling it ‘one of the most thrilling demos in TED history’, according to Miroculus. To see and hear why this opinion is accurate, and how Miriam will work in concert with a smartphone camera and cloud interface, I urge you to check out the ~11 minute TEDGlobal presentation at this link, which also gives a short, layperson introduction to miRNA biomarkers in blood for cancer.

Oh, One More Thing

Taken from graymatters.com

Taken from graymatters.com

Although this post focuses on nucleic acids, it’s worth noting that protein biomarkers in blood are also being investigated. In view of increased awareness and media attention about concussion injuries in the National Football League (NFL), a timely example of protein biomarkers for diagnosis of chronic traumatic encephalopathy (CTE)—which heretofore has not been possible by any test—is in development.

Currently the only way to diagnose CTE is through a post-mortem autopsy, but Aethlon Medical Inc. intends to change that with the diagnostic test being developed by its subsidiary Exosome Sciences. The test being studied is designed to identify an abnormal protein called tau that builds up in brain tissue as a result of repetitive head trauma. CTE researchers believe that they have developed a means of measuring plasma exosomal tau. Researchers thought that exosomes had potential as a means of identifying CTE because they cross the blood-brain barrier and can provide a unique method of measuring certain aspects of the contents of brain cells through a blood test.

Exosome Science was able to use its diagnostic blood test in 78 NFL players with histories of concussions, as well as in a control group made up of 16 athletes involved in non-contact sports. The subjects are all part of a much larger NIH-funded project called DETECT, which is focused on developing a variety of biomarkers for CTE and involves researchers at Boston University School of Medicine and the University of Washington.

Look for a future post here about DETECT involving nucleic acid biomarkers.

As always, your comments are welcomed.

Genes in Space

  • High School Student’s PCR Experiments Launched to Space Station
  • Program Evaluates Epigenetics Linked to Astronaut’s Altered Immunity
  • Amplyus Has Big Plans for Its Tiny, Low-Cost PCR Device

In my 2013 blog post on the 30th anniversary of the invention of Nobel Prize-winning PCR by Kary Mullis, I ventured to say that PCR of DNA or RNA was the most widely used—and enabling—method for all life sciences on planet Earth. This accolade can now be expanded to extraterrestrial space in view of PCR experiments to be carried out in the International Space Station (ISS) following the April 8th launch from Kennedy Space Center in Florida aboard NASA’s Cargo Resupply Services flight (CRS-8).

Taken from earthkam.org

Taken from earthkam.org

What makes this “out of this world” milestone for PCR even more exciting is that it’s the result of competition among students in high school—yes, high school—to conceive and design PCR-based studies relevant to living in space. Following is a brief synopsis of the program, the winning high school student, and a small startup company with big plans for its low-cost miniPCR™ device.
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World’s Smallest Real-Time PCR Device

  • Fits in the Palm of your Hand, and Has Single-Molecule Sensitivity
  • Analyzes 4 Samples, and Can be Modified to do 8 
  • Project Leader Reveals Commercialization Details 

I think we’re all fascinated by catchy headlines touting the world’s biggest, tallest, etc., so a recent publication by Ahrberg et al. in venerable Lab on a Chip claiming the world’s smallest real-time PCR device instantly struck me as blogworthy. It also seemed quite apropos as a follow-up to my previous blogs on the continuing shrinkage, so to speak, of real-time PCR technology for point-of-care qPCR diagnostics or other emerging applications in the field.

This hand-held real-time PCR device, developed by A*STAR Singapore, is amazingly small in comparison to the first real-time PCR system introduced by Applied Biosystems in 1995 that weighed 350 pounds and had a width of 7 feet, thus requiring an entire bench top.

pcr

Left: World’s smallest real-time PCR device. (Taken from Ahrberg et al). Right: Applied Biosystems 7700 real-time PCR system. (Taken from distrobio.com).

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Norovirus: Science Behind the Headline

  • The Virus is Quite Common with 267 Million Cases and 200,000 Deaths Annually
  • RT-PCR is the Detection Method of Choice
  • First Cell-Culture System May Speed Drug and Vaccine Development

We’ve all seen TV news stories about disgruntled passengers disembarking cruise ships returning to port early because of an outbreak of nasty gastroenteritis (i.e. inflammation of the stomach and intestines leading to nausea, vomiting, diarrhea, and stomach cramps). Norovirus (NoV) is the causative agent of these frequently reoccurring “nightmare” cruises, of which 13 have been reported since 2012, sickening some 200-600 passengers. It’s not just limited to cruises, the virus affected 100+ students at a school in Eugene, Oregon last year. And now there’s new evidence for transmission of NoV by eating oysters—which I will therefore not eat in the future.

Taken from counselheal.com.

Taken from counselheal.com.

But perhaps the most NoV-related media attention—and investor ire or litigant action—has been recently focused on gastroenteritis outbreaks at Chipotle—a popular restaurant chain. A criminal investigation is under way at Chipotle, and according to an Associated Press report the company has been served with a federal subpoena.

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‘Genospirituality’—In the Spirit of the Holidays

This last blog for 2015 comes at a time when many of us are looking forward to the upcoming holiday season to enjoy, in various ways, being or sharing with family and friends, and reflecting thankfully for what we have. We often refer to this as getting into “the spirit” of the holidays, regardless of one’s religious or secular beliefs.

In this context, and with the nucleic acids research-relatedness of my blogs in mind, I thought it would be apropos to tell you a bit about some intriguing research aimed at assessing genes associated with spirituality, by which is meant “an inner search for enlightenment achieved through practices such as prayer [religious] or meditation [secular]”, as elaborated elsewhere.

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DNA Barcodes to Follow Food and Foil Fraud

  • Fork-to-Farm Traceability Proven Practical
  • Uses qPCR Decoding of DNA Barcode “tags”
  • Consumer Concerns May Curb Commercialization

In earlier blogs here I’ve highlighted how sequencing of DNA barcodes has been used to identify fraudulent food sales—such as cheap meat or fish passed off as expensive steak or sashimi—and the same for mislabeled herbal products. Now, I’d like to share with you some nifty work by researchers who have applied qPCR to detect DNA barcode “tags” to follow foodstuff from your fork back to the farm (or farms) from which it originated.

This is not a simple goal to achieve, scientifically, and would certainly have been exceedingly useful, societally, in tracing the origin of food-related incidents such as listeria-contaminated cantaloupes that led to 29 deaths several years ago. In fact, just a few weeks ago there was a deadly outbreak of salmonella linked to cucumbers imported from Mexico wherein 4 people died and 732 were sickened by the bacteria.

They all look the same, but sometimes it’s very importatnt to find out from where they came. Taken from en.wikipedia

They all look the same, but sometimes it’s very important to find out where they came from. Taken from en.wikipedia

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Point-of-Care PCR 2.0

  • Ubiquitome Quickens Pace of POC Apps for Its Freedom4
  • Cepheid Unveils its POC Diagnostics System
  • Hopkins Crew Brews “Coffee Mug-Sized” Gizmo for Fully Automated Chlamydia Testing
Kiwi Dr. Jo-Ann Stanton holding Ubiquitome’s Freedom4 at Tri-Con 2015

Kiwi Dr. Jo-Ann Stanton holding Ubiquitome’s Freedom4 at Tri-Con 2015

Regular readers of this blog will recall a recent byline exclaiming “Honey I Shrunk the qPCR Machine”, which spotlighted the unveiling of startup company Ubiquitome’s first point-of-care (POC) product—Freedom4—developed in New Zealand. Up until then, this far away—for me—exotic island country brought to mind folks fondly nicknamed Kiwi—after the native flightless bird, not Chinese fruit. Mightily impressed by this tiny but powerful qPCR device, I vowed to thereafter keep an eye on these Kiwis’ democratized POC apps enabled by its nifty handheld 4-sample high-performance qPCR device.

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Gene-Expression Biomarkers Can Detect Depression

  • First-ever Lab Test for Depression Found Using RT-PCR
  • FDA Approval as Diagnostic Possible by Early 2016
  • Huge Potential Market as 1-in-10 US Adults Suffer from Depression

While it’s normal for everyone to occasionally feel blue or sad, prolonged bouts of depression that interfere with normal life are indicative of a serious mental health issue. While there are numerous forms and differing severity of depressive disorders, as described at a National Institute of Mental Health (NIMH) website, only two factual aspects of this illness really stand out in my opinion:

Redder countries have higher depression rates. Bluer countries have lower depression rates. Taken from The Washington Post.

Redder countries have higher depression rates. Bluer countries have lower depression rates. Taken from The Washington Post.

  • Depression is a very common illness. The Centers for Disease Control and Prevention estimates that 1-in-10 US adults suffer from depression, which reportedly costs close to $50B annual in lost productivity in the work place. Globally, more than 350 million people of all ages are afflicted with depression, according to recent statistics from the World Health Organization (WHO). By the year 2020, WHO estimates that depression will be the second leading cause of “lost years of healthy life”, following heart disease. Incidentally, as seen from the map below, depression rates around the world vary significantly among countries.
  • Depression is diagnosed based on the patients’ self-report of their symptoms and the evaluation of one or more structured psychiatric interviews with the patient by a psychiatrist, psychologist or primary care physician. The absence of direct, non-subjective measures of depression can lead to relatively lengthy time-to-treatment, non-reporting, or—sorry to say—fraudulent claims and/or treatments based solely on what is said as opposed to what is objectively measured.

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Top Picks from Tri-Con 2015

  • “Honey I Shrunk the qPCR Machine” Tops Presentations
  • High School Student Wins Popular Vote for Best Poster
  • BioFire Defense FilmArray is More Interesting Exhibitor
  • Extra Bonus: Swimming with the Sharks

The 22nd International Molecular Medicine Tri-Conference—better known as Tri-Con—took place on Feb 15-20 in San Francisco, where I and 3,000+ other attendees from over 40 countries took part in a jam-packed agenda. In this blog I’ll briefly share my top 3 picks—and an “extra bonus”—but first some insights into the challenges involved in navigating a large conference like this.

The first challenge was scoping out four simultaneously occurring “channels”—diagnostics, clinical, informatics, and cancer—to select as many interesting items as possible from all the presentations (500), panel discussions (30), posters (150), and free “lunch-nars.” The new Tri-Con’15 app with a word and name-searchable agenda (including abstracts) made this easier than previous years. I was even able to put selected items into a calendar/to-do list with 15-min reminder alarms—very slick and convenient. Every big conference should have an app like this!

The second challenge came once I was physically onsite. It took a bit of effort to navigate from one room to another in the huge, multi-room Moscone Center without GPS guidance. I was also struggling to make it to the talks and events on time without getting hijacked by bumping into friends—which happened a lot.

The third and final challenge had to do with posters. Given all of the other exciting options during the conference, I really had to focus to stay on-task and make sure I was present at my poster at the specified times, yet alone try to get around to the other posters of interest. This was definitely not easy, since my poster entitled Pushing the Limits of PCR, qPCR and RT-PCR Using CleanAmp™ Hot Start dNTPs attracted a steady stream of interested visitors. But that’s a great challenge to have, so I can’t complain too much.

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Children at Risk from Deadly Respiratory Virus EV-D68

  • Frightening Statistics From CDC
  • CDC Updated U.S. Map of Outbreak & Advice of What to be Aware
  • CDC Develops Rapid Real-Time RT-PCR Test for Detection
  • Some Speculate on Linking Outbreak to the “Southern Border Invasion”

Ebola virus is dominating news reports lately, and perhaps rightly so considering the worldwide impact. Turning our attention, however, to actual incidents of infection and death in the U.S., enterovirus (EV) D68 poses a much greater threat and warrants our attention—especially if you or your friends have young children.

On September 24, Eli Waller’s parents were worried that their 4 year-old son had pink eye and kept him home from school so that he wouldn’t infect other children. He seemed otherwise healthy. What happened next was shocking.

Eli Waller (Credit Andy Waller, via Associated Press). Taken from NY Times.

Eli Waller (Credit Andy Waller, via Associated Press). Taken from NY Times.

‘He was asymptomatic and fine, and the next morning he had passed,’ said Jeffrey Plunkett, the township’s health officer. ‘The onset was very rapid and very sudden,’ quoted the NY Times.

A week later the Centers for Disease Control and Prevention (CDC) confirmed that Eli had been infected with EV-D68.

EV-D68 was seen as early as August of this year as hospitals in Missouri and Illinois reported increased visits from children with respiratory illness. Soon, the virus was identified in 43 states and detected in 594 patients, 5 of which died.

After reading this very sad—if not frightening—story, I decided to research EV-D68 for this “hot topic” blog, which I’m dedicating to little Eli Waller.

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