Proof-of-concept study may eventually lead to new clinical laboratory urine tests for fast, non-invasive detection of cancer
Here is the latest example of researchers finding useful biomarkers in urine for diagnosing certain cancers. The discovery comes from the University of Michigan Health Rogel Cancer Center, where, in a proof-of-concept study, scientists developed a urine-based test that screens for circulating free DNA (cfDNA) fragments (aka, cell-free DNA) released by tumors in the head and neck. If they confirm these findings, it’s possible the technology could be adapted into a non-invasive clinical laboratory test for selected cancers.
One such cancer is human papillomavirus (HPV) which, though “widely recognized for causing cervical cancer” is “increasingly found to cause cancers in the mouth, throat, and other head and neck regions,” according to a U-M Medical School press release.
The U-M study findings could lead to an early, non-invasive test for the detection of cancer, as compared to traditional urine or blood-based liquid biopsy testing.
“In this study, we provide evidence to support the hypothesis that conventional assays do not detect ultrashort fragments found in urine since they are designed to support longer DNA fragments. Our team used an unconventional approach to develop a urine test for HPV-positive head and neck cancer ctDNA detection,” said Chandan Bhambhani, PhD (above), Research Lab Specialist Intermediate at University of Michigan and co-first author of the study, in a news release. Clinical laboratories may soon have a new urine-based test for detecting cancer. (Photo copyright: LinkedIn.)
According to the researchers, benefits of urine testing include:
Testing with urine is convenient for people who may be unable to access healthcare and phlebotomy services.
Urine has low biohazard risk and may be easily collected in large amounts, compared with blood.
Ongoing collection of urine could make way for TR-ctDNA “kinetics to be used as a high time-resolution biomarker” to monitor patients’ response to treatment.
However, urine, the researchers cautioned, must be analyzed in a different manner if it is to be comparable in efficiency to blood-based ctDNA testing.
“There have been mixed reports on the efficiency of TR-ctDNA detection compared with that of blood ctDNA. A potentially crucial factor for the analysis of TR-ctDNA is knowing the length of TR-ctDNA fragments present in urine, because this affects assay design for optimal sensitivity in TR-ctDNA detection,” the researchers explained.
New Assay Detects Ultrashort DNA Fragments
To complete their study, the U-M researchers developed an ultrashort HPV droplet digital PCR (polymerase chain reaction) assay that enabled detection of TR-ctDNA from HPV-associated oropharyngeal squamous cell carcinoma (HPV OPSCC), BioTechniques reported.
The assay was made to target the HPV16 E6 (Human papillomavirus 16) gene and to measure TR-ctDNA in patients with HPV OPSCC, the JCI Insight paper noted.
“The HPV16 E6 gene represents a highly recurrent ctDNA target in the population of patients with HPV OPSCC,” the researchers wrote in JCI Insight, adding:
Targeting ultrashort fragments was essential “for robust TR-ctDNA detection.”
Results in urine with patients with HPV OPSCC was consistent with results from plasma ctDNA.
The test, still in the discovery phase, was mailed to patients who were being treated for the disease and who reside within 100 miles of Ann Arbor, Mich. They returned urine samples for testing at the U-M lab and to get insights into possible post-treatment needs.
“Using longitudinal urine samples from a small case series, we showed proof of concept for early detection of cancer recurrence. Thus, our results indicate that by targeting ultrashort DNA fragments, TR-ctDNA becomes a viable approach for HPV OPSCC detection and potentially for cancer recurrence monitoring after treatment,” the authors wrote.
Further Studies, Possible Test Expansion
HPV infection—and especially HPV type 16—is a growing risk factor for oropharyngeal cancers, according to the National Cancer Institute.
The U-M Rogel Cancer Center scientists plan more studies to leverage the information urine may carry about an individual’s health. The researchers intend to expand the scope of their new test to other cancers including breast cancer and acute myeloid leukemia.
“The test that has been developed has detected cancer far earlier than would typically happen based on clinical imaging. As such, these promising results have given us the confidence to broaden the scope of this study, seeking to expanding distribution even further,” said J. Chad Brenner, PhD, Associate Professor of Otolaryngology-Head and Neck Surgery, U-M Medicine, and co-senior author of the study, in the news release.
The University of Michigan Health study exemplifies scientists’ commitment to new categories of biomarkers that can be used for medical laboratory tests and prescription drugs. And by focusing on urine, the researchers made it possible for patients to collect specimens themselves and send them to the medical laboratory for analysis and reporting.
Results of an earlier study in which locks of Beethoven’s hair underwent genetic analysis showed the composer ‘had a predisposition for liver disease and became infected with hepatitis B’
Here is an example of modern technologies being used with “historical biospecimens” to solve long-standing mysteries or questions about the illnesses of famous historical figures. Clinical laboratory scientists at the Mayo Clinic have used modern-day chemical analysis techniques to answer a 200-year-old question: What caused Ludwig van Beethoven’s deafness and other health problems?
“Such lead levels are commonly associated with gastrointestinal and renal ailments and decreased hearing but are not considered high enough to be the sole cause of death,” the authors wrote.
Beethoven’s death at age 56 has been attributed to kidney and liver disease, CNN reported. Even if the lead concentrations were not the sole cause, they would nevertheless be regarded as lead poisoning, lead study author Nader Rifai, PhD, told CNN.
“If you walk into any emergency room in the United States with these levels, you will be admitted immediately and you will undergo chelation therapy,” he said.
“It is believed that Beethoven died from liver and kidney disease at age 56. But the process of understanding what caused his many health problems has been a much more complicated puzzle, one that even Beethoven himself hoped doctors could eventually solve,” CNN reported, adding, “The composer expressed his wish that his ailments be studied and shared so ‘as far as possible at least the world will be reconciled to me after my death.’” Mayo clinical laboratory scientists are using chemical analysis on authenticated locks of Beethoven’s hair to do just that. (Photo copyright: Joseph Karl Stieler/Public Domain.)
Mass Spectrometry Analysis
Mayo Clinic’s metals laboratory, led by chemist Paul Jannetto, PhD, an associate professor in the Department of Laboratory Medicine and Pathology and Laboratory Director at the Mayo Clinic, performed the analysis on two authenticated locks of Beethoven’s hair, using inductively coupled plasma mass spectrometers.
The researchers found that one lock had 258 micrograms of lead/gram and the other had 380 micrograms. Normally they would expect to find less than four micrograms.
“These are the highest values in hair I’ve ever seen,” Jannetto told The New York Times. “We get samples from around the world and these values are an order of magnitude higher.”
The researchers also found that the composer’s hair had four times the normal level of mercury and 13 times the normal amount of arsenic.
Rifai and other researchers noted that Beethoven drank large amounts of plumbed wine, and at the time it was common to sweeten wine with lead acetate, CNN reported.
The composer also could have been exposed to lead in glassware. He likely absorbed high levels of arsenic and mercury by eating fish caught from the Danube River in Vienna.
David Eaton, PhD, a toxicologist, pharmacologist, and Professor Emeritus, Department of Environmental and Occupational Health Sciences at the University of Washington, told The New York Times that high levels of lead could have impaired Beethoven’s hearing through their effect on the nervous system. Additionally, he said the composer’s gastrointestinal ailments “are completely consistent with lead poisoning.”
Rifai told CNN that he’d like to study locks of hair from other 19th century Vienna residents to see how their lead levels compared with Beethoven’s.
Beethoven’s Genome and Genetic Predisposition for Liver Disease
Additional research published in May built on an earlier genomic analysis of Beethoven’s hair, which appeared in March 2023 in the journal Current Biology.
The international team included geneticists, archeologists, and immunologists who analyzed eight locks of hair attributed to the composer. They determined that five were authentic. One, known as the Stumpff Lock, appeared to be the best preserved. They used this lock to sequence Beethoven’s DNA.
“Although we could not identify a genetic explanation for Beethoven’s hearing disorder or gastrointestinal problems, we found that Beethoven had a genetic predisposition for liver disease,” the authors wrote. “Metagenomic analyses revealed furthermore that Beethoven had a hepatitis B infection during at least the months prior to his death. Together with the genetic predisposition and his broadly accepted alcohol consumption, these present plausible explanations for Beethoven’s severe liver disease, which culminated in his death.”
One surprising discovery was the likelihood of an extramarital affair on the composer’s father’s side, CNN reported. The researchers learned this in part by comparing his genetic profile with those of living relatives.
“Through the combination of DNA data and archival documents, we were able to observe a discrepancy between Ludwig van Beethoven’s legal and biological genealogy,” study coauthor Maarten Larmuseau, PhD, told CNN. Larmuseau is assistant professor, Faculty of Medicine, and head of the Laboratory of Human Genetic Genealogy at KU Leuven in Belgium.
The Mayo Clinic team used two locks authenticated in the 2023 study—the Bermann Lock and Halm-Thayer Lock—to perform their chemical analysis, CNN reported.
Beethoven’s Wishes
The earlier study noted that Beethoven wanted his health problems to be made public. In 1802, he wrote a document known as the Heiligenstadt Testament in which he asked that his physician, surgeon/ophthalmologist Johann Adam Schmidt, MD, discuss his disease after he died.
“For almost two years I have ceased to attend any social functions, just because I find it impossible to say to people: I am deaf,” Beethoven wrote at age 30, The New York Times reported. “If I had any other profession, I might be able to cope with my infirmity, but in my profession, it is a terrible handicap. And if my enemies, of whom I have a fair number, were to hear about it, what would they say?”
The authors of the Current Biology paper wrote, “Genomic sequence data from authenticated locks of Beethoven’s hair provide Beethoven studies with a novel primary source, already revealing several significant findings relating to Beethoven’s health and genealogy, including substantial heritable risk for liver disease, infection with HBV [Hepatitis B], and EPP [extra pair paternity]. This dataset additionally permits numerous future lines of scientific inquiry.
“The further development of bioinformatics methods for risk stratification and continued progress in medical genetic research will allow more precise assessments both for Beethoven’s disease risk and for the genetic inference of additional phenotypes of interest.
“This study illustrates the contribution and further potential of genomic data as a novel primary source in historical biography,” the scientists concluded.
The work of the clinical laboratory professionals at Mayo Clinic also demonstrates how advances in various diagnostic technologies can enable pathologists and lab scientists to participate in solving long-standing health questions about historical figures, especially if their hair or other types of specimens survived and can be used in the analysis.
The ASBMB story notes that nanopore technology depends on differences in charges on either side of the membrane to force DNA or RNA through the hole. This is one reason why proteins pose such a challenge.
“Think of a cell as a miniature city, with proteins as its inhabitants. Each protein-resident has a unique identity, its own characteristics, and function. If there was a database cataloging the fingerprints, job profiles, and talents of the city’s inhabitants, such a database would undoubtedly be invaluable!” said Behzad Mehrafrooz, PhD (above), Graduate Research Assistant at University of Illinois at Urbana-Champaign in an article he penned for the university website. This research should be of interest to the many clinical laboratories that do protein testing. (Photo copyright: University of Illinois.)
How the Maglia Process Works
In a Groningen University news story, Maglia said protein is “like cooked spaghetti. These long strands want to be disorganized. They do not want to be pushed through this tiny hole.”
His technique, developed in collaboration with researchers at the University of Rome Tor Vergata, uses electrically charged ions to drag the protein through the hole.
“We didn’t know whether the flow would be strong enough,” Maglia stated in the news story. “Furthermore, these ions want to move both ways, but by attaching a lot of charge on the nanopore itself, we were able to make it directional.”
The researchers tested the technology on what Maglia described as a “difficult protein” with many negative charges that would tend to make it resistant to flow.
“Previously, only easy-to-thread proteins were analyzed,” he said in the news story. “But we gave ourselves one of the most difficult proteins as a test. And it worked!”
Maglia now says that he intends to commercialize the technology through a new startup called Portal Biotech.
Detecting Post-Translational Modifications in the UK
In another recent study, researchers at the University of Oxford reported that they have adapted nanopore technology to detect post-translational modifications (PTMs) in protein chains. The term refers to changes made to proteins after they have been transcribed from DNA, explained an Oxford news story.
“The ability to pinpoint and identify post-translational modifications and other protein variations at the single-molecule level holds immense promise for advancing our understanding of cellular functions and molecular interactions,” said contributing author Hagan Bayley, PhD, Professor of Chemical Biology at University of Oxford, in the news story. “It may also open new avenues for personalized medicine, diagnostics, and therapeutic interventions.”
Bayley is the founder of Oxford Nanopore Technologies, a genetic sequencing company in the UK that develops and markets nanopore sequencing products.
The news story notes that the new technique could be integrated into existing nanopore sequencing devices. “This could facilitate point-of-care diagnostics, enabling the personalized detection of specific protein variants associated with diseases including cancer and neurodegenerative disorders,” the story states.
In another recent study, researchers at the University of Washington reported that they have developed their own method for protein sequencing with nanopore technology.
“This opens up the possibility for barcode sequencing at the protein level for highly multiplexed assays, PTM monitoring, and protein identification!” Motone wrote.
Single-cell proteomics, enabled by nanopore protein sequencing technology, “could provide higher sensitivity and wider throughput, digital quantification, and novel data modalities compared to the current gold standard of protein MS [mass spectrometry],” they wrote. “The accessibility of these tools to a broader range of researchers and clinicians is also expected to increase with simpler instrumentation, less expertise needed, and lower costs.”
There are approximately 20,000 human genes. However, there are many more proteins. Thus, there is strong interest in understanding the human proteome and the role it plays in health and disease.
Technology that makes protein testing faster, more accurate, and less costly—especially with a handheld analyzer—would be a boon to the study of proteomics. And it would give clinical laboratories new diagnostic tools and bring some of that testing to point-of-care settings like doctor’s offices.
Best of all, the researchers say the test could provide an inexpensive means of early diagnosis. This assay could also be used to monitor how well patients respond to cancer therapy, according to a news release.
The protein had previously been identified as a promising biomarker and is readily detectable in tumor tissue, they wrote. However, it is found in extremely low concentrations in blood plasma and is “well below detection limits of conventional clinical laboratory methods,” they noted.
To overcome that obstacle, they employed an ultra-sensitive immunoassay known as a Simoa (Single-Molecule Array), an immunoassay platform for measuring fluid biomarkers.
“We were shocked by how well this test worked in detecting the biomarker’s expression across cancer types,” said lead study author gastroenterologist Martin Taylor, MD, PhD, Instructor in Pathology, Massachusetts General Hospital and Harvard Medical School, in the press release. “It’s created more questions for us to explore and sparked interest among collaborators across many institutions.”
“We’ve known since the 1980s that transposable elements were active in some cancers, and nearly 10 years ago we reported that ORF1p was a pervasive cancer biomarker, but, until now, we haven’t had the ability to detect it in blood tests,” said pathologist and study co-author Kathleen Burns, MD, PhD (above), Chair of the Department of Pathology at Dana-Farber Cancer Institute and a Professor of Pathology at Harvard Medical School, in a press release. “Having a technology capable of detecting ORF1p in blood opens so many possibilities for clinical applications.” Clinical laboratories may soon have a new blood test to detect multiple types of cancer. (Photo copyright: Dana-Farber Cancer Institute.)
Simoa’s Advantages
In their press release, the researchers described ORF1p as “a hallmark of many cancers, particularly p53-deficient epithelial cancers,” a category that includes lung, breast, prostate, uterine, pancreatic, and head and neck cancers in addition to the cancers noted above.
“Pervasive expression of ORF1p in carcinomas, and the lack of expression in normal tissues, makes ORF1p unlike other protein biomarkers which have normal expression levels,” Taylor said in the press release. “This unique biology makes it highly specific.”
Simoa was developed at the laboratory of study co-author David R. Walt, PhD, the Hansjörg Wyss Professor of Bioinspired Engineering at Harvard Medical School, and Professor of Pathology at Harvard Medical School and Brigham and Women’s Hospital.
The Simoa technology “enables 100- to 1,000-fold improvements in sensitivity over conventional enzyme-linked immunosorbent assay (ELISA) techniques, thus opening the window to measuring proteins at concentrations that have never been detected before in various biological fluids such as plasma or saliva,” according to the Walt Lab website.
Simoa assays take less than two hours to run and require less than $3 in consumables. They are “simple to perform, scalable, and have clinical-grade coefficients of variation,” the researchers wrote.
Study Results
Using the first generation of the ORF1p Simoa assay, the researchers tested blood samples of patients with a variety of cancers along with 406 individuals, regarded as healthy, who served as controls. The test proved to be most effective among patients with colorectal and ovarian cancer, finding detectable levels of ORF1p in 58% of former and 71% of the latter. Detectable levels were found in patients with advanced-stage as well as early-stage disease, the researchers wrote in Cancer Discovery.
Among the 406 healthy controls, the test found detectable levels of ORF1p in only five. However, the control with the highest detectable levels, regarded as healthy when donating blood, “was six months later found to have prostate cancer and 19 months later found to have lymphoma,” the researchers wrote.
They later reengineered the Simoa assay to increase its sensitivity, resulting in improved detection of the protein in blood samples from patients with colorectal, gastroesophageal, ovarian, uterine, and breast cancers.
The researchers also employed the test on samples from 19 patients with gastroesophageal cancer to gauge its utility for monitoring therapeutic response. Although this was a small sample, they found that among 13 patients who had responded to therapy, “circulating ORF1p dropped to undetectable levels at follow-up sampling.”
“More Work to Be Done”
The Simoa assay has limitations, the researchers acknowledged. It doesn’t identify the location of cancers, and it “isn’t successful in identifying all cancers and their subtypes,” the press release stated, adding that the test will likely be used in conjunction with other early-detection approaches. The researchers also said they want to gauge the test’s accuracy in larger cohorts.
“The test is very specific, but it doesn’t tell us enough information to be used in a vacuum,” Walt said in the news release. “It’s exciting to see the early success of this ultrasensitive assessment tool, but there is more work to be done.”
More studies will be needed to valid these findings. That this promising new multi-cancer immunoassay is based on a clinical laboratory blood sample means its less invasive and less painful for patients. It’s a good example of an assay that takes a proteomic approach looking for protein cancer biomarkers rather than the genetic approach looking for molecular DNA/RNA biomarkers of cancer.
Scientists believe useful new clinical laboratory assays could be developed by better understanding the huge number of ‘poorly researched’ genes and the proteins they build
Researchers have added a new “-ome” to the long list of -omes. The new -ome is the “unknome.” This is significant for clinical laboratory managers because it is part of an investigative effort to better understand the substantial number of genes, and the proteins they build, that have been understudied and of which little is known about their full function.
The Unknome Database includes “thousands of understudied proteins encoded by genes in the human genome, whose existence is known but whose functions are mostly not,” according to a news release.
The database, which is available to the public and which can be customized by the user, “ranks proteins based on how little is known about them,” the PLOS Biology paper notes.
It should be of interest to pathologists and clinical laboratory scientists. The fruit of this research may identify additional biomarkers useful in diagnosis and for guiding decisions on how to treat patients.
“These uncharacterized genes have not deserved their neglect,” said Sean Munro, PhD (above), MRC Laboratory of Molecular Biology in Cambridge, England, in a press release. “Our database provides a powerful, versatile and efficient platform to identify and select important genes of unknown function for analysis, thereby accelerating the closure of the gap in biological knowledge that the unknome represents.” Clinical laboratory scientists may find the Unknome Database intriguing and useful. (Photo copyright: Royal Society.)
Risk of Ignoring Understudied Proteins
Proteomics (the study of proteins) is a rapidly advancing area of clinical laboratory testing. As genetic scientists learn more about proteins and their functions, diagnostics companies use that information to develop new assays. But did you know that researchers tend to focus on only a small fraction of the total number of protein-coding DNA sequences contained in the human genome?
The study of proteomics is primarily interested in the part of the genome that “contains instructions for building proteins … [which] are essential for development, growth, and reproduction across the entire body,” according to Scientific American. These are all protein-coding genes.
Proteomics estimates that there are more than two million proteins in the human body, which are coded for 20,000 to 25,000 genes, according to All the Science.
To build their database, the MRC researchers ranked the “unknome” proteins by how little is known about their functions in cellular processes. When they tested the database, they found some of these less-researched proteins important to biological functions such as development and stress resistance.
“The role of thousands of human proteins remains unclear and yet research tends to focus on those that are already well understood,” said Sean Munro, PhD, MRC Laboratory of Molecular Biology in Cambridge, England, in the news release. “To help address this we created an Unknome database that ranks proteins based on how little is known about them, and then performed functional screens on a selection of these mystery proteins to demonstrate how ignorance can drive biological discovery.”
In the paper, they acknowledged the human genome encodes about 20,000 proteins, and that the application of transcriptomics and proteomics has “confirmed that most of these new proteins are expressed, and the function of many of them has been identified.
“However,” the authors added, “despite over 20 years of extensive effort, there are also many others that still have no known function.”
They also recognized limited resources for research and that a preference for “relative safety” and “well-established fields” are likely holding back discoveries.
The researchers note “significant” risks to continually ignoring unexplored proteins, which may have roles in cell processes, serve as targets for therapies, and be associated with diseases as well as being “eminently druggable,” Genetic Engineering News reported.
Setting up the Unknome Database
To develop the Unknome Database, the researchers first turned to what has already come to fruition. They gave each protein in the human genome a “knownness” score based on review of existing information about “function, conservation across species, subcellular localization, and other factors,” Interesting Engineering reported.
It turns out, 3,000 groups of proteins (805 with a human protein) scored zero, “showing there’s still much to learn within the human genome,” Science News stated, adding that the Unknome Database catalogues more than 13,000 protein groups and nearly two million proteins.
The researchers then tested the database by using it to determine what could be learned about 260 “mystery” genes in humans that are also present in Drosophila (small fruit flies).
“We used the Unknome Database to select 260 genes that appeared both highly conserved and particularly poorly understood, and then applied functional assays in whole animals that would be impractical at genome-wide scale,” the researchers wrote in PLOS Biology.
“We initially selected all genes that had a knownness score of ≤1.0 and are conserved in both humans and flies, as well as being present in at least 80% of available metazoan genome sequences. … After testing for viability, the nonessential genes were then screened with a panel of quantitative assays designed to reveal potential roles in a wide range of biological functions,” they added.
“Our screen in whole organisms reveals that, despite several decades of extensive genetic screens in Drosophila, there are many genes with essential roles that have eluded characterization,” the researchers conclude.
Clinical Laboratory Testing Using the Unknome Database
Future use of the Unknome Database may involve CRISPR technology to explore functions of unknown genes, according to the PLOS Biology paper.
Munro told Science News the research team may work with other research efforts aimed at understanding “mysterious proteins,” such as the Understudied Proteins Initiative.
The Unknome Database’s ability to be customized by others means researchers can create their own “knownness” scores as it applies to their studies. Thus, the database could be a resource in studies of treatments or medications to fight diseases, Chemistry World noted.
According to a statement prepared for Healthcare Dive by SomaLogic, a Boulder, Colorado-based protein biomarker company, diagnostic tests that measure proteins can be applied to diseases and conditions such as:
“The 27-protein model has potential as a ‘universal’ surrogate end point for cardiovascular risk,” the researchers wrote in Science Translational Medicine.
Proteomics definitely has its place in clinical laboratory testing. The development of MRC-LMB’s Unknome Database will help researchers’ increase their knowledge about the functions of more proteins which should in turn lead to new diagnostic assays for labs.
