Proteins in human saliva make up its proteome and may be the key to new, precision medicine diagnostics that would give clinical pathologists new capabilities to identify disease
Clinical pathologists may soon have an array of new precision medicine diagnostic tools based on peoples’ saliva. There are an increasing number of “–omes” that can be the source of useful diagnostic biomarkers for developing clinical laboratory tests. The latest is the world’s first saliva protein biome wiki.
Called the Human Salivary Proteome Wiki (HSP Wiki), the “public data platform,” which was created by researchers at the University of Buffalo, is the “first of its kind,” according to Labroots, and “contains data on the many thousands of proteins present in saliva.”
The HSP Wiki brings together data from independent studies on proteins present in human saliva. One of the researchers’ goals is to speed up the development of saliva-based diagnostics and personalized medicine tools.
In “The Human Salivary Proteome Wiki: A Community-Driven Research Platform,” published in the Journal of Dental Research, the researchers wrote, “Saliva has become an attractive body fluid for on-site, remote, and real-time monitoring of oral and systemic health. At the same time, the scientific community needs a saliva-centered information platform that keeps pace with the rapid accumulation of new data and knowledge by annotating, refining, and updating the salivary proteome catalog.
“We developed the Human Salivary Proteome (HSP) Wiki as a public data platform for researching and retrieving custom-curated data and knowledge on the saliva proteome. … The HSP Wiki will pave the way for harnessing the full potential of the salivary proteome for diagnosis, risk prediction, therapy of oral and systemic diseases, and preparedness for emerging infectious diseases,” they concluded.
“This community-based data and knowledge base will pave the way to harness the full potential of the salivary proteome for diagnosis, risk prediction, and therapy for oral and systemic diseases, and increase preparedness for future emerging diseases and pandemics,” Stefan Ruhl, DDS, PhD (above right, with Omer Gokcumen, PhD, Associate Professor of Biological Sciences on left), Professor, Department of Oral Biology, University of Buffalo, and lead researcher of the study, told Labroots. Development of precision medicine clinical laboratory diagnostics is part of their research goals. (Photo copyright: University of Buffalo.)
Where Does Saliva Come From?
Saliva is a complex biological fluid that has long been linked to oral health and the health of the upper gastrointestinal tract. Only recently, though, have scientists begun to understand from where in the body saliva proteins originate.
The authors wrote: “Salivary proteins are essential for maintaining health in the oral cavity and proximal digestive tract, and they serve as potential diagnostic markers for monitoring human health and disease. However, their precise organ origins remain unclear.
“Through transcriptomic analysis of major adult and fetal salivary glands and integration with the saliva proteome, the blood plasma proteome, and transcriptomes of 28+ organs, we link human saliva proteins to their source, identify salivary-gland-specific genes, and uncover fetal- and adult-specific gene repertoires,” they added.
“Our results pave the way for future investigations into glandular biology and pathology, as well as saliva’s use as a diagnostic fluid,” the researchers concluded.
Saliva plays a crucial role in digestion by breaking down starches. It also provides a protective barrier in the mouth. When salivary glands malfunction, patients can face serious health consequences. Although clinicians and scientists have long understood the importance of saliva to good health, the question now is whether it contains markers of specific diseases.
“The Human Salivary Proteome Wiki contains proteomic, genomic, transcriptomic data, as well as data on the glycome, sugar molecules present on salivary glycoproteins. New data goes through an interdisciplinary team of curators, which ensures that all input data is accurate and scientifically sound,” noted Labroots.
The graphic above “shows the interconnectedness of the thousands of salivary proteins originating from blood plasma, parotid glands, and submandibular and sublingual glands. The diagram is one of many tools available to researchers and clinicians through the Human Salivary Proteome Wiki,” noted a UBNow blog post. (Graphic copyright: University of Buffalo.)
Omics and Their Role in Clinical Laboratory Diagnostics
Proteomics is just one of several hotly-researched -omics that hold the potential to develop into important personalized medicine and diagnostics tools for pathologists. Genomics is a related area of research being studied for its potential to benefit precision medicine diagnostics.
However, unlike genomes, which do not change, proteomes change constantly. That is one of the main reasons studying the human salivary proteome could lead to valuable diagnostics tools.
Combining the study of the -omes with tools like mass spectrometry, a new era of pathology may be evolving. “With the rapid decrease in the costs of omics technologies over the past few years, whole-proteome profiling from tissue slides has become more accessible to diagnostic labs as a means of characterization of global protein expression patterns to evaluate the pathophysiology of diseases,” noted Pathology News.
Saliva and the Age of Precision Medicine
The study of the -omes may be an important element in the evolution of precision medicine, because of its ability to provide information about what is happening in patients’ bodies at the point of care.
Thus, a full understanding of the proteome of saliva and what causes it to change in response to different health conditions and diseases could open the door to an entirely new branch of diagnostics and laboratory medicine. It is easy and non-invasive to gather and, given that saliva contains so much information, it offers an avenue of study that may improve patients’ lives.
It also would bring us closer to the age of precision medicine where clinical laboratory scientists and pathologists can contribute even more value to referring physicians and their patients.
Scientists
at St. Jude’s have discovered that performing different genetic tests on pediatric
cancer patients, and then combining those test results, may help guide and
improve patient care.
The research was part of a St. Jude’s project called Genomes
for Kids (G4K), a study to determine how genetic information may be used to
diagnose and treat pediatric cancers.
Through this project, the researchers hope to learn why tumors form in
children and predict how tumors will respond to certain treatments.
‘It’s
a Whole Lot of Sequencing.’
Few tragedies are worse than cancer in children. This is where precision medicine treatments can be critical, and multiomics may play an important role in the development of new therapies.
Multiomics refers to a biological analysis approach in which
multiple “omes” are analyzed together in a collaborative way to locate relevant
biomarkers and functional relationships. These “omes” include:
To perform their research, the St. Jude scientists examined
253 pediatric cancer patients by conducting whole genome
sequencing (WGS), whole
exome sequencing (WES), and RNA
sequencing of their tumors. They also looked at the WGS and WES of
non-cancerous tissues extracted from the same cancer patients.
“It is a whole lot of sequencing. I admit that,” Scott Newman, PhD,
Group Lead, Bioinformatics Analysis at St. Jude’s, told The
Scientist.
“With results available in a clinically relevant time frame, and pricing becoming increasingly comparable to the radiology and pathology tests, WGS is becoming more accessible to pediatric oncology patients,” said Scott Newman, PhD (above), Group Lead, Bioinformatics Analysis, at St. Jude’s, in an American Society of Human Genetics (ASHG) news release. (Photo copyright: ASHG.)
As a result of their three-platform testing, the researchers
discovered there was at least one finding for each patient that could be useful
in providing a diagnosis, revealing risks for individual patients, or
pinpointing which drugs may be most beneficial for a particular patient in
nearly 200 (79%) of the cases. Such findings are at the heart of precision
medicine.
The researchers also compared their sequencing results to
cancer panels that use next-generation
sequencing (NGS) to target specific genes or mutations relevant to a
certain cancer phenotype.
During this portion of the research, they discovered that the cancer panels
missed 11% to 16% of actionable genes relating to diagnosis, prognosis, and
treatment.
“This is either good news or bad news, depending on how you
look at it,” Newman said. “Personally, I am amazed at how well these panels do
and how well they have been designed. But, if you want to know every mutation
that you would probably want to report, you have to do comprehensive
sequencing.”
First Multi-Platform Genomic Sequencing Study
“To
our knowledge, this is the first clinical study where this comprehensive three-platform
genomic sequencing approach was offered prospectively to all pediatric oncology
patients,” said Kim Nichols, MD,
Director, Division of Cancer Predisposition at St. Jude’s, in a St.
Jude’s blog post.
The testing costs $8,600 per patient, but is considered worth
it to improve patient diagnosis, prognosis, and treatment for pediatric cancer
patients.
“Compared with the cost of many
other procedures that children with cancer undergo, the cost is likely
comparable, or even less—for example, compared with complex surgical procedures
or multiple radiology tests,” Nichols said.
In addition, the test results are available in less than 30
days, which makes them more valuable, as time can be a critical asset to cancer
management.
The scientists hope this type of three-platform genetic
testing can help guide care for pediatric cancer patients.
“Because
so few of the molecular lesions in pediatric cancer are targetable by specific
drugs, currently it is the diagnostic and prognostic insights provided by the
three-platform approach that appear most clinically impactful,” said Nichols.
“From a diagnostic perspective, tumors may look the same under a microscope,
but the identification of specific genetic changes can direct you to the correct
diagnosis, and therefore, the most appropriate therapy. From a prognostic
perspective, you will have different risk stratifications depending on results.”
The results of the research were presented at the 2018
annual meeting of the American Society of Human Genetics in San Diego last
October. The St. Jude’s researchers hope that this type of research can drive
wider adoption of WGS in the assessment of pediatric tumors to improve patient
outcomes. Pathologists and medical laboratory scientists will want to watch for
additional research findings as the team at St. Jude’s uses this approach on
more pediatric cancer patients.
These “off-target” genetic alterations demonstrate that certain CRISPR base editors need further refinement in a research finding of interest to pathologists
Could CRISPR
DNA-editing technology unintentionally effect RNA as well? A new study conducted
at Massachusetts General Hospital
(MGH) suggests that it can. Clinical
laboratories doing genetic testing will want to understand why this
research implies that refinements to CRISPR may be needed for it to be accurate
in therapeutic applications.
For years, a huge value of CRISPR (Clustered Regularly
Interspaced Short Palindromic Repeats) base editors have been their ability to
edit genes or convert a specific DNA base without breaking the DNA. Now, the MGH
scientists have discovered that certain CRISPR base editors may extend beyond
the targeted DNA and perform unwanted edits to RNA, according to a news release.
“Most investigation of off-target base editing has focused
on DNA, but we have found that this technology can induce large numbers of RNA
alterations as well. This surprising finding suggests the need to look at more
than just genetic alterations when considering unintended off-target effects of
base editors in cells,” J. Keith Joung, MD,
PhD, MGH Pathologist and Professor of Pathology at Harvard Medical School, stated in the news release.
The MGH scientists published their study in Nature.
How the MGH Researchers
Found Off-Target Effects on RNA
The researchers had set their sights on developing a base
editor that targets cytosine,
according to the study.
“Previous studies of cytosine base editor specifically have
identified off-target DNA edits in human cells. Here, we show that a cytosine
base editor with rat APOBEC1
[rAPOBEC1] enzyme can cause extensive transcriptome-wide RNA
cytosine deamination in
human cells,” the scientists wrote in Nature.
According to the news
release, when the researchers put base editors into human liver and kidney cells,
they found their technology induced efficient edits at the target DNA site.
However, they also discovered tens of thousands of cytosine-to-uracil edits in the cells. They
found that deaminases, an enzyme that acts as a catalyst, which they used in
their base editor to change DNA, also altered the RNA in the cells, Science reported.
“Base editors are still incredibly powerful tools. This is just another parameter we need to understand,” J. Keith Joung, MD, PhD (above), MGH Pathologist and Professor of Pathology at Harvard Medical School, told Science. (Photo copyright: Massachusetts General Hospital.)
The researchers developed a way to reduce the unwanted RNA
edits, while maintaining the targeted DNA effects. They came up with cytosine
base editor variants, which they dubbed SElective Curbing of Unwanted RNA
Editing (SECURE).
“We engineered two cytosine base editor variants bearing
rAPOBEC1 mutations that substantially decreased the number of RNA edits in
human cells,” the researchers wrote in their study.
However, they also
called for changes to how base editors are used. “For research applications,
scientists using base editors will need to account for potential RNA off-target
effects in their experiments,” the MGH news release notes. “For therapeutic
applications, our results further argue for limiting the duration of base-editor
expression to the shortest length of time possible and the importance of
minimizing and accounting for potential impacts of these effects in safety
assessments.”
Other Studies Explore CRISPR
Other studies published earlier this year on mice and on rice also suggested
that “modified CRISPR-Cas9 technology will need to be further refined before it
can safely be used for research and therapeutic applications,” The Scientist reported.
Clinical laboratory leaders and pathologists recognize
CRISPR technology is changing the way research is done for diagnosing disease
as well as guiding treatment. Dark Daily has reported on key
CRISPR developments over many years.
And now, though the MGH study may appear to be a set-back
for CRISPR, it also may propel further research into possible therapeutic
applications of CRISPR base editing. It’s a development worth watching.
Methods that target the causes of acidity could become part of precision medicine cancer treatments and therapies
Researchers at Massachusetts
Institute of Technology (MIT) have found that acidic environments enable
tumor cells to strengthen through protein production. And that when acidic surfaces
extend beyond a tumor’s interior, and come into contact with healthy tissue,
cancer can spread.
The results of their study will interest anatomic
pathologists who review tissue biopsies to diagnose cancer and help identify
the most effective therapies for cancer patients. Currently, there are no new clinical
laboratory tests under development based on MIT’s research.
The researchers published their findings in the journal Cancer Research. Their paper also
shared how tumor acidity can be identified and reversed.
“Our findings reinforce the view that tumor acidification is an important driver of aggressive tumor phenotypes, and it indicates that methods that target this acidity could be of value therapeutically,” noted Frank Gertler, PhD (above), in a news release. Gertler is an MIT Professor of Biology, a member of MIT’s Koch Institute for Integrative Cancer Research, and a Senior Author of the study. (Photo copyright: MIT News.)
Acidity is a Tumor Cell’s
Friend
Acidity results from lack of oxygen in tumors and enables
tumor cell growth. “Acidification of the microenvironment plays established roles
in tumor progression and provides a hostile milieu that advantages tumor cell
survival and growth compared to non-cancerous cells,” the researchers wrote in Cancer Research.
In their study, the MIT scientists sought to learn:
What areas of a tumor are actually acidic?
How does acidosis propel cells to
invade surrounding healthy tissues?
They used a nanotechnology platform
called pHLIP (pH Low
Insertion Peptide) to sense pH at the surface of cancer cells and then insert a
molecular probe into the cell membranes. “This brings nanomaterial to close
proximity of cellular membrane,” noted a research study
conducted at the University of Rhode Island by scientists who developed the
pHLIP technology.
Medical News Today reported that the MIT scientists
used pHLIP to map the acidity in human breast cancer tumors implanted in mice.
When it detected a cell in an acidic environment, pHLIP sent a small protein
molecule into the cell’s membrane. The scientists found that acidosis was not
confined to the oxygen-rich tumor core. It extended to the stroma, an important boundary
between healthy tissue and malignant tumor cells.
“We characterized the spatial characteristics of acidic
tumor microenvironments using pHLIP technology, and demonstrated that
tumor-stroma interfaces are acidic, and that cells within the acidic front are
invasive and proliferative,” the scientists wrote in Cancer Research.
What Stimulates
Acidity and How to Reverse It?
The MIT researchers sought the reasons, beyond hypoxia, for
high acidity in tumor tissue.
“There was a great deal of tumor tissue that did not have
any hallmarks of hypoxia that was quite clearly exposed to acidosis. We started
looking at that, and we realized hypoxia probably wouldn’t explain the majority
of regions of the tumor that were acidic,” Gertler pointed out in the MIT news
release.
So what did explain it? The researchers pointed to aerobic
glycolysis, a “condition in which glucose is converted to lactate in the presence
of oxygen,” according to an article published by StatPearls. “Cancer
stem cells (CSC) within a tumor are notorious for aerobic glycolysis. Thus,
extensive aerobic glycolysis has been indicative of aggressive cancer,” the
paper’s authors noted.
During their study, the MIT scientists found:
Cells at the tumor surface shifted to aerobic
glycolysis, “a type of metabolism that generates lactic acid, making way
for high acidity,” and
“Tumor acidosis gives rise to the expression of molecules
involved in cell invasion and migration. This reprogramming, which is an
intracellular response to a drop in extracellular pH, gives the cancer cells
the ability to survive under low-pH conditions and proliferate,” said Nazanin Rohani, PhD, former
postdoctoral researcher in the MIT Koch Institute for Integrative Cancer
Research, and Lead Author of the study, in the news release.
Could a Reduction in Acidity Reverse Tumor Growth?
In another experiment, the researchers fed sodium
bicarbonate (baking soda) to mice with breast or lung tumors. The tumors became
less acidic and metastatic.
“It adds to the sense that this pH dynamic is not permanent.
It’s reversible. I think that’s an important addition to an ongoing discussion
about the role of pH in tumor behavior,” said Ian Robey, PhD, in an
MITblog
post. Robey is a Research Assistant Professor, Department of Medicine
at the University of Arizona, and Full Investigator at the Arizona Cancer Center. He was not
involved in the MIT research.
Spreading the Word on
How Cancer Spreads
The MIT study is important—not only to anatomic pathologists—but
also to oncologists and cancer patients worldwide. Cancer is not simple to
diagnose and treat. The MIT study may provide important insights into targeting
cancer care and precision
medicine treatments.
Even in its early stages the Human Cell Atlas project is impacting the direction of research and development of RNA sequencing and other genetic tests
No one knows exactly how many cell types exist in the human body. Though traditional texts place numbers in the hundreds, recent studies have found ranges from thousands to tens of thousands. Anatomic pathologists and clinical laboratory scientists know that the discovery of new types of human cells could lead to the creation of new medical laboratory tests.
So, it’s an important development that leaders of the Human Cell Atlas Consortium, a project comparable to the Human Genome Project, have set out to determine the exact numbers of cell types. And their findings could open up an entirely new field of diagnostic testing for clinical laboratories and anatomic pathology and lead to advances in precision medicine.
With the ability to identify cell types and sub-types associated with human disease and health conditions, medical labs could have a useful new way to help physicians make diagnoses and select appropriate therapies.
Begun in 2016, the group’s mission according to the Human Cell Atlas website is “To create comprehensive reference maps of all human cells—the fundamental units of life—as a basis for both understanding human health and diagnosing, monitoring, and treating disease.”
The ambitious project aims to catalog every cell type in the human body and “account for and better understand every cell type and sub-type, and how they interact.”
Striving for Deeper Understanding of the Basics
Cells are the basic building blocks of life, but scientists don’t know exactly how many different types of cells there are.
In an NPR interview, Aviv Regev, PhD, Professor of Biology and a core member at the Broad Institute of MIT and Harvard, investigator at the Howard Hughes Medical Institute, and co-leader of the Human Cell Atlas Consortium, said, “No one really knows how many [cells types] there will be,” adding, “People guess anything from the thousands to the tens of thousands. I’m not guessing. I would rather actually get the measurements done and have a precise answer.”
In an innovative move, Regev and her team improved the method they were already using to sort cells—single-cell RNA sequencing. “All of sudden we moved from something that was very laborious—and we could do maybe a few dozen or a few hundred—to something where we could do many, many thousands in a 15- to 20-minute experiment,” she told NPR.
But the project is massive. A typical human body contains about 37.2 trillion cells. So, the Human Cell Atlas scientists decided to complete preliminary pilot projects to identify the most efficient and effective strategies for sampling and analyzing the various cells to create the full atlas.
“It’s kind of like we’re trying to find out what are all the different colors of Lego building blocks that we have in our bodies,” Sarah Teichmann, PhD, Head of Cellular Genetics and Senior Group Leader at Wellcome Sanger Institute in the UK, and co-leader of the Human Cell Atlas Consortium, told NPR. “We’re trying to find out how those building blocks—how those Lego parts—fit together in three dimensions within each tissue.”
Sarah Teichmann, PhD (left), and Aviv Regev, PhD (right), are co-leaders of the Human Cell Atlas Consortium, an ambitious project of MIT/Harvard Broad Institute that seeks to “create comprehensive reference maps of all human cells—the fundamental units of life—as a basis for both understanding human health and diagnosing, monitoring, and treating disease.” Such an advance could lead to significant advances in clinical laboratory and pathology testing and move healthcare closer to true precision medicine. (Photo copyrights: University of Cambridge and MIT/Broad Institute.
Some of the early pilot projects include a partnership with the Immunological Genome Project (ImmGen) to study and map the cells in the immune system. According to the Human Cell Atlas website, the partnership “will combine:
“deep knowledge of immunological lineages;
“clinical expertise and infrastructure needed to procure and process diverse samples;
“genomic and computational expertise to resolve the hundreds of finely differentiated cell types that compose all facets of the immune system; and,
the genomic signatures that define them.”
Other areas the pilot projects will address include:
the Human Developmental Cell Atlas (HDCS), which will investigate the highly specialized cells involved in human development.
Progress So Far
In the two short years since the Human Cell Atlas project began much work has already been accomplished, according to a news release. In addition to organizing the consortium and obtaining funding, the collaborators have published a white paper describing their goals and a framework for reaching them, as well as launching the pilot projects.
Such an ambitious project, however, is not without barriers and challenges. Regev and Teichmann, along with other collaborators, outlined some of those challenges in an article published in Nature.
The complexity of the human body combined with rapidly changing technology make simply agreeing on the scope of the project challenging. In order to meet that particular challenge, the collaborators plan to work in phases and drafts, which will allow for some flexibility and increasing focus on specifics as they go.
Other challenges include:
keeping the entire project open and fair;
procuring samples with consent and in an appropriate manner; and,
organizing in an efficient and effective manner.
The collaborators have developed and detailed strategies for meeting each of these challenges.
The Human Cell Atlas could impact treatments for every disease that affects humans and bring healthcare closer to accomplishing precision medicine goals. By knowing what cells exist in what parts of the human body—and how they typically behave at their most basic levels—the MIT/Harvard/Broad Institute scientists hope to understand what’s happening when those cells “misbehave” in expected ways. The knowledge garnered from the Human Cell Atlas is likely to be invaluable to anatomic pathologists and clinical laboratories.
