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.

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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Liquid Biopsies Are Viewed as “Liquid Gold” for Diagnostics

  • Invasive Needles and Scalpels Seen as Passé
  • Noninvasive Sampling Advocates Focusing on Circulating Tumor Cells (CTCs) 
  • New Companies are Pursuing the Liquid Biopsy “Gold Rush”

Biopsy Basics

Ultrasound is a real-time procedure that makes it possible to follow the motion of the biopsy needle as it moves through the breast tissue to the region of concern, as discussed elsewhere (taken from oncopathology.info via Bing Images).

Ultrasound is a real-time procedure that makes it possible to follow the motion of the biopsy needle as it moves through the breast tissue to the region of concern, as discussed elsewhere (taken from oncopathology.info via Bing Images).

As defined in Wikipedia, a biopsy is ‘a medical test commonly performed by a surgeon or an interventional radiologist involving sampling of cells or tissues for examination.’ Biopsies can be excisional (removal of a lump or area), incisional (removal of only a sample of tissue), or a needle aspiration (tissue or fluid removal). Despite the value of these traditional types of biopsies, they are more or less invasive, lack applicability in certain instances, and require accurately “going to the source” of concern, as pictured to the right, for ultrasound-guided breast cancer biopsy. Better methodology is highly desirable and is the topic of this post. By the way, if you want to peruse a lengthy list of scary risks associated with various type of common invasive biopsies, click here to see what I found in Google Scholar by searching “incidence of complications from biopsies.”

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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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PCR Better than Pap Test for Preventing Cervical Cancer

“Power of PCR” as a Transformative Diagnostic Method

  • FDA Approves Roche PCR Test for Cervical Cancer Screening
  • Automated Test Replaces Pap Test as First-line Cervical Cancer Screening
  • Demonstrates the “Power of PCR” as a Transformative Diagnostic Method

Pap Test

The Papanicolaou test—aka Pap test, Pap smear, cervical smear, or smear test—is a method of cervical screening used to detect potentially pre-cancerous and cancerous processes in the endocervical canal of the female reproductive system. Unusual findings are often followed up by more sensitive diagnostic procedures, and, if warranted, interventions that aim to prevent progression to cervical cancer.

Proper interpretation of microscopic results requires a “trained eye.” This is evident from the representative example shown below, which I found in an online educational textbook and, quite frankly, had trouble discerning the visual keys described in the verbatim caption. Notwithstanding this issue, Pap tests were—until now—the accepted “gold standard.”

pap

Taken from homepage.smc.edu via Bing Images.

The source document for this Pap smear reads as follows. “The cytologic features of normal squamous epithelial cells can be seen at the center top and bottom, with orange to pale blue plate-like squamous cells that have small pyknotic nuclei. The dysplastic cells in the center extending to upper right are smaller overall with darker, more irregular nuclei.”

The eponymous test was pioneered by Georgios Papanikolao, a prominent Greek doctor, who in 1928 was the first to report that uterine cancer could be diagnosed by means of a vaginal smear. However, the importance of this work was not widely recognized until his 1943 publication of Diagnosis of Uterine Cancer by the Vaginal Smear, coauthored by Herbert F. Traut, both at Cornell University Medical College.

ResearchGeorgios Papanicolaou moved to Miami, Florida in 1961 to establish the Papanicolaou Cancer Research Institute at the University of Miami, but died in 1962 prior to its opening. Papanicolaou was the recipient of the Albert Lasker Award for Clinical Medical Research in 1950—this award is sometimes referred to as “America’s Nobels,” as eighty-six Lasker laureates have received the Nobel Prize. Papanikolaou’s portrait appeared on the Greek 10,000-drachma banknote of 1995-2001, prior to its replacement by the Euro.

Cervical Cancer Statistics

Cervical cancer is the second most common cancer in women worldwide, according to an NIH publication in 2007. Country-by-country data for cervical cancer reveal a striking geographical distribution. According to currently available U.S. Centers for Disease Control (CDC) FastStats, cervical cancer mortality in the U.S. in 2010 was ~4,000 or ~2.5 deaths per 100,000 females.

The global statistics provided by Cancer Research U.K. are far more saddening. Worldwide there were more than ~275,000 deaths from cervical cancer in 2010 that accounted for ~10% of female cancer deaths.

Remarkably, mortality rates are reported to vary seventeen-fold between the different regions of the world. By estimating the years-of-life-lost (YLL) by young and middle-aged women (25-64 years old) in different regions of the world, YLL attributed to cervical cancer is the most important cause of YLL for all cancers in Latin America, the Caribbean, and populous regions of Sub-Saharan Africa and South-Central Asia. The overall picture is not very sensitive to the age-weighting function used. The report notes that, since this loss of life is preventable using existing technologies, more health-resource allocation in low income settings is needed.

Pap Test Statistics

Currently available CDC FastStats for Pap test use in the U.S. in 2010 (the most recent year available) are as follows:

  • Percent of women 18 years of age and over who had a Pap test within the past 3 years: 73.2%
  • Number of physician office visits during which Pap tests were ordered or provided: 29.4 million
  • Number of hospital outpatient department visits during which Pap tests were ordered or provided: 2.4 million

Pap Test Recommendations as of 2013

To put today’s blog-post headline about switching from Pap to PCR in perspective, here are snippets from the most recent CDC guidelines and comments made available in a January 2013 press release headlined with “more women getting Pap tests as recommended [but] some women get Pap tests without need.”

  • In 2012, the U.S. Preventive Services Task Force, American College of Obstetricians and Gynecologists and American Cancer Society recommended that women, beginning at age 21, should start Pap test screening every three years.
  • The same groups agree that screening is unnecessary for most women who have had a total hysterectomy (removal of the uterus and uterine cervix) for non-cancerous reasons, or for women aged 65 years and older with several years of normal test results.
  • Studies analyzed Pap test survey data from CDC’s Behavior Risk Factor Surveillance System found the following:
    • The percentage of women aged 18-21 years who reported never being screened increased from 23.6% in 2000 to 47.5% in 2010; however, screening is not recommended for women under the age of 21.
    • In 2010, 58.7% of women aged 30 years and older who had a hysterectomy were still given a Pap test.
    • Because of the Affordable Care Act (aka Obamacare), many private health plans and Medicare now cover certain preventive services, including cervical cancer screening, with no copays or other out-of-pocket costs.

HPV: The Cervical Cancer-Causing Agent and Key to Early Detection

In a landmark publication in 1999 entitled Human papillomavirus is a necessary cause of invasive cervical cancer worldwide, Dutch investigators used PCR data to establish that the worldwide HPV prevalence in cervical carcinomas is 99.7 per cent. They noted that “the presence of HPV in virtually all cervical cancers implies the highest worldwide attributable fraction so far reported for a specific cause of any major human cancer.” More importantly, they presciently concluded that “the extreme rarity of HPV-negative cancers reinforces the rationale for HPV testing in addition to, or even instead of, cervical cytology in routine cervical screening.”

Due in part to technical challenges posed by numerous genotypes of HPV with varying cancer causality detailed elsewhere, and unavoidable time-consuming clinical studies required for FDA approval, it has taken ~15 years for a PCR test to now be poised to displace the Pap test as the primary diagnostic approach for early detection of cervical cancer.

Those of you who are interested in the technical underpinnings of Roche’s investigations to this end are referred to this 2013 publication by Roche and collaborators entitled Development and characterization of the cobas human papillomavirus test. In contrast to the tedious Pap test protocol and its “visually challenging” manual microscopic analysis, this “cobas”-based PCR test provided by Roche is fully automated.  The test process involves two instruments: one that completes sample preparation (COBAS® AmpliPrep) and another that performs the PCR process and detection of the pathogen DNA in real time (COBAS® TaqMan® Analyzer).

Incidentally, I traced-back the term “cobas” to late 1970’s Roche instrumentation named the “cobas-bio” analyzer, but could not decipher what “cobas” stands for! If any of you know the answer, please let us know by a comment at the end of this post.

FDA Panel Recommends Replacement for the Pap Test

This attention-grabbing headline of a March 2014 NY Times article by Andrew Pollock was the catalyst for my decision to research and write this blog exemplifying the “power of PCR” as a transformative diagnostic method. While this and numerous other popular news media all made reference to an FDA panel’s report, it took some digging to find the actual source-report, which is an 80-page pdf that can be accessed here to peruse in detail, if you wish. However, a much shorter but essential-fact-laden article by Joyce Frieden, News Editor of MedPage Today provided the following excerpts.

The FDA’s Medical Devices Advisory Committee Microbiology Panel agreed by a vote of 13-0 in each of three successive votes that the cobas® viral DNA test for HPV—made by Roche Molecular Systems—was safe and effective for cervical cancer screening, and that the benefits of the tests outweighed the risks. The Panel recommended that this Roche HPV test replace the Pap smear as the first-line standard of care for cancer screening.

The Roche test is seen as better than Pap tests in finding precancerous lesions (taken from the NY Times).

The Roche test is seen as better than Pap tests in finding precancerous lesions (taken from the NY Times)

The cobas® test currently has approval as a follow-up assessment for women 21 and older who have abnormal Pap tests, and as a co-test with the Pap smear to screen for the high-risk p16 and p18 HPV strains in women 30 to 65. The test comprises genotyping for HPV16 and 18 and pooled assessment of 12 additional high-risk HPV strains.

According to the proposal submitted by Roche, women 25 and older who test positive for HPV16 or 18 would proceed directly to colposcopy for further assessment.

Patients who test negative for HPV16 or 18 but positive for the other high-risk strains would have a Pap test to determine the need for colposcopy. Women who have a completely negative test would be followed at their physician’s discretion.

Panelists did express some concerns about dropping the age at which women should have the test from 30 to 25. The ATHENA study of over 47,000 patients with long-term follow-up used as the basis for the application found that about 11% of women ages 25 to 29 tested positive for HPV16 or 18 with the cobas test, compared with 7.28% among women 25 to 29 who had cytology alone as their first-line screening. Panel member Paula Hillard, MD, of Stanford University in California, was quoted as saying that would mean more patients in that age group “will be anxious about potentially having cancer.”

In addition, Hillard is quoted as expressing concern about off-label use. “I’m concerned that all those women potentially with other high-risk positivity won’t go to Paps next but go [straight] to colposcopy. That’s not what’s proposed here, but what control does FDA have once it’s out there?”

Panelist Kenneth Noller, MD, of the American Board of Obstetrics and Gynecology, in Dallas, agreed that real-world use could differ from the protocol proposed by Roche. He’s quoted as saying that “I’ve been watching how people practice; if you’re high-risk HPV positive you’re going to get colposcopy.” Furthermore, he said “that doesn’t necessarily mean it’s bad—it’s what you do with the colposcopy.”

Noller added that although he was “somewhat biased against dropping the age to 25 before I came here … I find the data presented today somewhat compelling to drop it to 25.”

Agreeing with this was panel member Kimberly Hanson, MD, MHS, of the University of Utah and ARUP Laboratories, both in Salt Lake City: “now we have the opportunity to identify women earlier, and to me that’s compelling,” adding that “although colposcopy is invasive and can be anxiety-provoking, it’s really very safe, so I think I’m leaning toward earlier screening.”

According to the summary submitted by FDA staff members, “The data show that the proposed primary screening indication for the cobas HPV test detects more women with disease and requires fewer women without disease to go to colposcopy than cytology alone.”

Benefit-risk analyses favored the HPV DNA test whether expressed in terms of number of cases of high-grade cervical disease per 10,000 women screened or per 100 colposcopy procedures.

The FDA is not bound to follow its advisory committees’ recommendations, but does so in most cases. On April 25—coincidentally DNA Day 2014—the FDA formally approved Roche’s HPV test as the First-Line Cervical Cancer Screening Method.

The “Entrenchment Factor”

At the risk of “throwing cold water” on the aforementioned PCR test benefits, I feel compelled to quote from Pollak’s NY Times story that ended with the following caveat.

“The Pap test, which is well entrenched and has been highly successful, will not go away quickly, if at all, however.

Assuming the FDA itself agrees with its advisory committee and approves the new use of Roche’s test, it would become just another option, not a replacement for the older testing regimens. And many doctors will not adopt the new test unless professional societies recommend it in guidelines, which could take years.”

Let’s all hope that these professional societies—and any other persuasive factors—lead to relatively rapid adoption by doctors.

As always, your comments are welcomed.

Nanomedicine: Using ‘Tiny Little FedEx Trucks’ to Target Breast Cancer Tumors

breastcancerlogo

As we enter October, national breast cancer awareness month, I thought it an appropriate time to review some of the latest advances in breast cancer research. Let’s start with a review of the latest statistics published by the NCI and a look at current treatments, then we’ll explore some of the most promising new research in this field.

According to the National Institutes of Health (NIH) National Cancer Institute (NCI) Cancer Topics for Breast Cancer, the projected statistics on breast cancer for women in the United States in 2013 are grim: 232,340 new cases and 39,620 deaths. Moreover, global statistics for breast cancer were estimated to be over 1.5 million new diagnoses and 500,000 deaths in 2010—with the majority of these being in low and middle income countries, which presents a major challenge of a different kind.

The NIH NCI website provides an informative online booklet entitled What You Need To Know About™ Breast Cancer to learn about breast cancer types, staging, treatment, and questions to ask the doctor. There is also NCI’s overview of Cancer Advances in Focus: Breast Cancer that provides the following perspectives for “today” and “tomorrow.”

Today

  • Among women diagnosed with breast cancer during the period from 1999 through 2006, 90% were expected to survive their disease at least 5 years. Among white women, the 5-year relative survival rate was 91%; among African American women, it was 78%. The increase in breast cancer survival seen since the mid-1970s has been attributed to both screening and improved treatment.
  • Breast-conserving surgery (lumpectomy) followed by local radiation therapy has replaced mastectomy as the preferred surgical approach for treating early-stage breast cancer.
  • Routine mammographic screening is an accepted standard for the early detection of breast cancer. The results of eight randomized trials, the NIH-ACS Breast Cancer Detection Demonstration Projects, and other research studies showed that mammographic screening can reduce the mortality from breast cancer.
  • Hormonal therapy with selective estrogen receptor modulators (SERMs), such as tamoxifen, and aromatase inhibitors is now standard in the treatment of women with estrogen receptor-positive breast cancer, both as adjuvant therapy and in the treatment of advanced disease. Estrogen receptor-positive breast cancer cells can be stimulated to grow by the hormone estrogen. SERMs interfere with this growth stimulation by preventing estrogen from binding to the estrogen receptor. In contrast, aromatase inhibitors block estrogen production by the body. Food and Drug Administration (FDA)-approved aromatase inhibitors include anastrozole, exemestane, and letrozole.
  • Tamoxifen and another SERM, raloxifene, have been approved by the FDA as treatments to reduce the risk of breast cancer in women who have an increased risk of developing the disease.
  • The monoclonal antibody trastuzumab is an accepted treatment for breast cancers that overproduce a protein called human epidermal growth factor receptor 2, or HER2. This protein is produced in abnormally high amounts by about 20% of breast tumors. Breast cancers that overproduce HER2 tend to be more aggressive and are more likely to recur. Trastuzumab targets the HER2 protein specifically, and this antibody, in conjunction with adjuvant chemotherapy, can lower the risk of recurrence of HER2-overproducing breast cancers by about 50% in comparison with chemotherapy alone.
  • Several breast cancer susceptibility genes have now been identified, including BRCA1, BRCA2, TP53, and PTEN/MMAC1. Approximately 60% of women with an inherited mutation in BRCA1 or BRCA2 will develop breast cancer sometime during their lives, compared with about 12% of women in the general population.

Tomorrow

  • We will use our rapidly increasing knowledge in the fields of cancer genomics and cell biology to develop more effective and less toxic treatments for breast cancer and to improve our ability to identify cancers that are more likely to recur. Moreover, we will use this knowledge to tailor breast cancer therapy to the individual patient.
  • We will use our increasing knowledge of the immune system to enhance the body’s ability to recognize and destroy cancer cells. The knowledge we have acquired thus far has facilitated the development of several promising breast cancer treatment vaccines that are currently under clinical evaluation.
  • We will use advanced technologies, including genomic technologies, to improve our ability to detect breast cancer at its earliest stages, when it is most treatable, and to better define individual risk for this disease.
  • We will strive to understand, address, and eliminate factors that contribute to the higher mortality from breast cancer experienced by African American women compared with women of other racial and ethnic groups.

Managing the Nation’s Cancer Research Portfolio

As a scientist and taxpayer, you’ll likely be very interested—as I was—in knowing much more about the specifics of what the NCI proposed for managing the Nation’s cancer research portfolio in 2013. This information is nicely laid out at an NCI website that includes a downloadable pdf version with programmatic details and a summary budget ($5.833 Billion).

ncir

National Cancer Institute Research Campus in Bethesda, MD (via Bing Images)

Among the details that I found particularly interesting is an introductory section called “Provocative Questions.” NCI launched an initiative late in 2010, seeking to go beyond the questions that are self-evident or that have been studied for many years. NCI “asked investigators to propose intriguing questions that need attention but might not otherwise get it or that have stumped us in the past but may be answered by new technologies. The initiative, which elicited a strong and exciting response from the research community, has recently funded its first 56 investigators.” In addition to a link therein for the Provocative Questions Project website, there is a link to a short video wherein NCI Director Harold Varmus, MD, discusses this project.

How do Normal v. Breast Cancer Cells Appear Microscopically?

Courtesy of NCI via Bing Images

Courtesy of NCI via Bing Images

From the descriptors given in the summary slide, provided by NCI, it is evident that breast cancer cell shape, size, and other properties reflect cellular dysfunctions, relative to normal cells, as is generally characteristic for other types of cancer. The accompanying high-resolution color enhanced scanning electron micrograph (SEM) of a breast cancer cell is visually stunning yet scary in view of the aforementioned death rate statistics.

A color enhanced scanning electron micrograph (SEM) of a breast cancer cell. Photograph: Science photo library (taken from theguardian.com via Bing Images)

A color enhanced scanning electron micrograph (SEM) of a breast cancer cell. Photograph: Science photo library (taken from theguardian.com via Bing Images)

Spotlight on an Expert

Prof. Esther H. Chang, MD PhD

Prof. Esther H. Chang, MD PhD

Prof. Esther H. Chang, MD PhD, is a member of the Departments of Oncology and Otolaryngology at the Lombardi Comprehensive Cancer Center of Georgetown University Medical Center. Before joining Georgetown University, Dr. Chang held positions at the National Cancer Institute (NCI), Stanford University, and the Uniformed Services University of Health Sciences. Currently, she is serving as the Interim President of the American Society for Nanomedicine and she is also an Executive Board Member of the International Society for Nanomedicine (ISNM) in Basel. Dr. Chang is the founding scientist of, as well as a Senior Consultant for, SynerGene Therapeutics, Inc. She has over 130 publications and has served as a member of a number of scientific advisory boards for NCI, NASA, the US Military Cancer Institute, and the Department of Energy.

I had the good fortune to become a collaborator of Dr. Esther H. Chang when she was working at the Uniformed Services University of Health Sciences, and I was literally across the street working at the FDA at NIH. We found that we shared a common interest in antisense therapeutics, which at that time (1980s) was a relatively new and—importantly—entirely novel drug development paradigm envisaged as affording more specific, less toxic anticancer agents. My lab could synthesize various chemically modified oligos, but had no molecular or cellular biology expertise. Dr. Chang’s lab at the time was working on Ras, which is a common oncogene in human cancer. We eventually published those initial collaborative investigations, and later reported a series of three papers on tumor-specific targeting and delivery of modified hybrid (DNA-RNA) anti-HER-2 siRNA analogs developed by TriLink (click here for details).

Dr. Chang’s achievements in specifically targeting tumors using antibody-fragment-tagged liposomal nanoparticles are indeed notable—and quotable—as outlined below.

“Tiny little FedEx trucks”

Tiny FedEx electric vehicles for delivery in Paris (Bing Images)

Tiny FedEx electric vehicles for delivery in Paris (Bing Images)

As scientists, we can appreciate the need to occasionally over simplify very complex technical concepts, and scientific jargon, in order to talk to nonscientists about our research. Consequently, I had to smile with appreciation when Dr. Chang referred to tumor cell-specific, targeted delivery of liposomal nanoparticles as involving “tiny little FedEx trucks” when interviewed on National Public Radio that can be listened to by clicking here.

Drug-carrying nanoparticles may be targeted to specific cell types. Illustration created by Amadeo Bachar for UCSD Center of Excellence in Nanomedicine; first published in Morachis et al., Pharm Rev 2012 (taken from Bing Images).

Dr. Chang’s use of this catchy metaphor is apropos for a couple of reasons. Just as trucks carry various types of cargo, liposomal nanoparticles (spheres pictured) can be loaded with various classes of therapeutic agents, ranging from traditional small-molecule entities to high-molecular weight genes, mRNAs, and mi/siRNA. Also, just as FedEx employs advanced technologies to locate ZIP-code intended-recipients, nanoparticles can utilize antibodies, fragments of antibodies (Y-shaped molecules pictured above) or other agents (ligands) for specific delivery to intended target cells via binding to cell surface receptors (pink molecules pictured above).

In an early publication entitled Tumor-targeted p53-gene therapy enhances the efficacy of conventional chemo/radiotherapy, Dr. Chang noted that a long-standing goal in gene therapy for cancer is a stable, low toxic, systemic gene delivery system that selectively targets tumor cells, including metastatic disease. In particular, ligand-directed tumor targeting of cationic liposome-DNA complexes (lipoplexes) showed promise for targeted gene delivery and systemic gene therapy. She demonstrated that Lipoplexes developed in her lab directed by ligands such as folate, transferrin or anti-transferrin receptor scFv antibody fragment, showed tumor-targeted gene delivery, expression, and anti-cancer effect in mouse models of human breast cancer, as well as prostate, head and neck cancers, seeming to meet these goals.

Based upon these promising results, this nanodelivery system has moved in human clinical trials. When I asked Dr. Chang about the status of such trials, and whether these results were applicable is some way to breast cancer, she offered the following comments:

“Tumor specificity is very crucial to a cancer therapy’s efficacy,” Chang asserts. “Because we have a targeting moiety, the nanocomplex will travel through the bloodstream and whenever it encounters a tumor cell — whether a primary tumor or metastasis — the nanoparticle will bind to it, enter the tumor cell and destroy it.”

This tumor targeted nanomedicine delivering the p53 gene (SGT-53) has already completed a Phase I safety trial, the results of which were described in an article entitled Phase I Study of a Systemically Delivered p53 Nanoparticle in Advanced Solid Tumors published in May of this year. The purpose of this trial was to give back to cancer patients the tumor-busting p53 gene they have lost.

She continued, “We were very encouraged by the results since the patients handled SGT-53 very well.  Only minimal side effects occurred. Even more exciting, at the end of treatment the majority of patients showed at least stable disease. We were also able to demonstrate targeted delivery of SGT-53 to metastatic cancer, and not to normal cells. These agents are intended to increase the effects of standard therapies by sensitizing the tumor cells to their killing effects. Thus, cancer is less likely to recur. We have seen fantastic anti-cancer responses in animal models with breast cancer, and we have seen this treatment working in various types of solid tumors in patients. Therefore, we think it is very likely that it will also be successful in treating breast cancer.”

Regarding future development of this agent, Chang stated “Three Phase II clinical trials are imminent in patients with pancreatic cancer, glioblastoma and lung cancer. We hope to evaluate this agent in patients with breast cancer in the near future.”

More on Nanomedicine for Cancer Therapy

Researchers in nanomedicine (a term first used in the late 1990s) aim to develop a variety of revolutionary tools:

  • Drug carriers that focus drug action at the site of disease to limit side effects.
  • Vaccines that are more feasible for use in areas with limited health care access (more stable, less expensive, and with minimal adverse reactions).
  • Imaging agents that produce signal detectable from significant depths only in diseased tissues.
  • Scaffolds for culturing engineered tissues that mimic the corresponding extracellular matrix and enable modulation of development over time and real-time monitoring of their activity and biochemistry.

As with any scientific field, its boundaries blur with those of related fields, including pharmaceutics, bio- and materials engineering, nuclear medicine, tissue engineering, and many others.

Schematic of localized magnetic delivery (taken from Nature Nano 2009 via Bing Images)

Schematic of localized magnetic delivery (taken from Nature Nano 2009 via Bing Images)

Magnetically Triggered, Local On-Demand Release of Drugs

Tran & Wilson highlight a particularly fascinating approach in nanomedicine aimed at making controllable magnetic drug delivery possible for the treatment of breast cancer. They note that a recent study published by Kong et al. in Nano Letters documents the synthesis and performance of porous silica nanocapsules filled with magnetic nanoparticles as a controllable magnetic drug delivery vector. Under a remotely applied radiofrequency magnetic field, these nanocapsules demonstrate on-off switchable release of the internally loaded drug payload. Both in vitro and in vivo studies using mouse breast cancer cell models demonstrate that the magnetic targeting of these nanocapsules allows for deep tumor penetration and subsequent on-demand release of the drug cargo, significantly reducing tumor cell viability.

“Chemical Antibodies” – Improving Nature with RNA Aptamers

Earlier this year, Shigdar et al. reported the use of sensitive “chemical antibodies” for diagnosis in breast cancer. They state that Epithelial cell adhesion molecule (EpCAM) is expressed at low levels in a variety of normal human epithelial tissues, but is overexpressed in 70–90% of carcinomas. From a clinico-pathological point of view, this has both prognostic and therapeutic significance. EpCAM was first suggested as a therapeutic target for the treatment of epithelial cancers in the 1990s. However, following several immunotherapy trials, the results have been mixed. It has been suggested that this is due, at least in part, to an unknown level of EpCAM expression in the tumors being targeted. Thus, selection of patients who would benefit from EpCAM immunotherapy by determining EpCAM status in the tumor biopsies is currently undergoing vigorous evaluation. However, current EpCAM antibodies are not robust enough to be able to detect EpCAM expression in all pathological tissues. Shigdar et al. go on to report a newly developed EpCAM RNA aptamer, also known as a chemical antibody, which is not only specific but also more sensitive than current antibodies for the detection of EpCAM in formalin-fixed paraffin-embedded (FFPE) primary breast cancers.

Schematic of dye- or drug-containing nanoparticle targeted to cancer cells by Y-shaped RNA aptamers (taken from Chemical & Engineering News 2012 via Bing Images)

Schematic of dye- or drug-containing nanoparticle targeted to cancer cells by Y-shaped RNA aptamers (taken from Chemical & Engineering News 2012 via Bing Images)

Twenty-mer aptamers were chemically synthesized with 2′-fluoro- or 2′-O-methyl-pyrimidine bases, a 3′-inverted deoxythymidine, and 5′-fluorescent tags. One of these aptamers showed no non-specific staining or cross-reactivity with tissues that do not express EpCAM. They were able to reliably detect target proteins in breast cancer xenograft where an anti-EpCAM antibody showed limited or no reactivity. They conclude that these results show the potential of aptamers in the future of histopathological diagnosis and as a tool to guide targeted immunotherapy.

I hope you leave this blog inspired by the promising developments that have been made in the field of breast cancer research and optimistic about the strides we, as a research community, continue to make toward finding a cure.

Breaking News:  FDA backs Roche drug as first-of-a-kind therapy to treat breast cancer before surgery

On September 12th, the Washington Post reported online that the FDA’s panel of cancer experts voted 13-0, with one abstention, that the benefits of Perjeta (a monoclonal antibody developed at Genentech) as an initial treatment for breast cancer outweigh its risks. The recommendation is not binding, but sets the stage for the FDA to clear the drug as the first pharmaceutical option approved to shrink or eliminate tumors before surgery. “We are supporting the movement of a highly active drug for metastatic breast cancer to the first-line setting, with the hope that women with earlier stages of breast cancer will live longer and better,” said Dr. Mikkael Sekeres, an associate professor of medicine at the Cleveland Clinic. Doctors hope that using cancer drugs earlier could help shrink tumors, making them easier to remove. In some cases, that could allow women to keep their breasts, rather than having a full mastectomy.

Perjeta works by blocking signals inside cancer cells that would tell them to divide and grow Photo: Roche (taken from itv.com via Bing Images).  Differences between HER family receptors are described here.

Perjeta works by blocking signals inside cancer cells that would tell them to divide and grow Photo: Roche (taken from itv.com via Bing Images). Differences between HER family receptors are described here.

Taken from Bing Images

Taken from Bing Images