Cancer arises from changes to genes. When exposed to chemicals, UV light, pathogens or other carcinogens, DNA can mutate, allowing cells to become malignant and invade other tissues. These changes get passed on to proteins that, along with tumor DNA, circulate in blood.
For many years, the medical and research communities have tried to use these proteins and DNA fragments as disease markers to find cancer early. Unfortunately, these approaches have not produced the diagnostic tests researchers anticipated. While scientists have had some success finding markers, such as the BRCA genes — which can identify women at increased risk for breast or ovarian tumors — these genetic changes by themselves cannot predict whether a patient will develop cancer.
But there may be other molecular markers researchers can pursue to detect cancer, including glycans — sugars attached to proteins. These complex carbohydrates help guide the formation and function of proteins. They also become altered by cancer. Could these be the markers scientists have been seeking?
The art of post-translational modification
When professor of analytical chemistry Carlito Lebrilla first started investigating glycans, the research field was sparse. Compared to studying proteins, glycans offered a far more difficult oad.
“Glycans are challenging because they’re not templated,” says Lebrilla. “For example, in DNA a gene is a template for RNA, which is a template for a protein. There are no specific genes for glycans; they’re added on afterward.”
This process, called post-translational modification, is kind of like adding aftermarket features to a car. After the changes are made, information about the make and model might not indicate how the car performs or even what it looks like.
Proteins also travel through an assembly line, in which enzymes called transferases attach glycans to them, sometimes hundreds. As many as 80 percent of all proteins go through this process. But if a sugar chain isn’t attached properly, the protein won’t function as it should. The hard part is figuring out how the process has gone awry.
Lebrilla’s team has adopted sophisticated methods to break glycans away from their proteins and analyze the fragments. They use a technique called mass spectrometry, which weighs molecules by determining how far a magnetic field can deflect them; heavier molecules just don’t go as far. These measurements are fed through a computer algorithm that compares the masses of these molecules to a library of known glycans. Given these sugars’ complexity, simply crunching the data is a huge task.
When Lebrilla first started working on glycans, mass spectrometry was mostly used to investigate proteins, but the technology lacked the finer definition needed to discriminate between glycans. In some cases, his lab had to devise new techniques to attain the resolution they needed.
“There aren’t many scientists who can do this work,” says Jay Solnick, a cancer researcher and professor in the Center for Comparative Medicine. “Carlito Lebrilla is one of the few people researching glycans.”
Sugars and disease
Understanding glycans could help researchers illuminate a number of critical biological processes. Lebrilla and colleagues are investigating glycans in breast milk and how they help infants establish beneficial gut bacteria. Also, because immune cells can be heavily glycosylated, his work could yield insights into autoimmune disorders, HIV and other conditions.
But the prize is cancer, and isolating glycan markers could help detect lung, prostate, gastric, ovarian and other tumors.
“We need better tests to detect cancer,” notes Lebrilla. “There are high false positive rates and unnecessary biopsies. We want to minimize these unneeded procedures.”
The Lebrilla lab scientists are collaborating with others, using their glycan expertise to detect specific cancers. They have been working closely with Solnick, who has been investigating the ramifications of Helicobacter pylori (H. pylori) infection, which affects 50 percent of the world’s population. H. pylori can cause ulcers, gastritis and gastric cancer, but determination of who will develop cancer is difficult. Finding a marker that indicates which H. pylori patients are at highest risk could improve care.
“There’s evidence that if you detect gastric cancer precursors early enough, you can treat the patient’s Helicobacter and prevent gastric cancer,” says Solnick.
Early research has been promising. Solnick, Lebrilla and colleagues recently published a paper that showed significant differences between glycan profiles for patients with gastritis and those with gastric cancer. This is good news, but it’s only a start.
“Right now we have statistical significance but not predictive value,” says Solnick. “If we can improve the predictability, we could create a diagnostic test with real clinical value.”
Finding ovarian cancer
Few people could benefit more from early detection than women with ovarian cancer. Like pancreatic cancer, ovarian cancer produces indistinct symptoms. Abdominal bloating and other gastrointestinal problems often lead physicians to investigate the intestinal tract rather than the pelvis.
“The biology of these cancers is such that by the time you figure out the patient has cancer, they have advanced disease, and it’s difficult to cure,” says Gary Leiserowitz, chief of the Division of Gynecologic Oncology.
Lebrilla’s research team had noted significant differences between the glycans in ovarian cancer cells and those in healthy cells. He contacted Leiserowitz, and they began looking for cancer markers.
Again, preliminary results have been promising. In recently published research, they tested samples from healthy women, patients with borderline ovarian tumors with minimal risk to become cancer, and patients with full-blown ovarian cancer. The glycan profiles distinguished patients among the three groups.
The first test, called a “training set,” measured different glycan expression in the serum samples and helped determine which sugars would help them differentiate between patient groups. The glycan-based biomarker panel developed with the training set distinguished women with ovarian cancer from healthy controls with 86 percent accuracy.
The researchers then conducted a “test set,” applying those measurements to entirely new patient samples. This also produced excellent results, detecting cancer with 70 percent accuracy, including both early- and late-stage cancers. The glycan markers distinguished between healthy and early-stage cancers as well as the standard diagnostic blood test for ovarian cancer, CA 125.
Because sample selection can bias results, they then swapped samples, creating a new training set with the patients from the previous testing set and vice versa. The results showed that the method works well, and that the developed markers are robust enough to be not overly influenced by patient selection.
Leiserowitz says the study is important because it replicates the real-life situations physicians often face, but he urges caution.
“Developing an actual diagnostic test is a long and tedious road,” he says. “Many studies look good at first but then aren’t reproducible. We are going through a very rigorous process to validate these results.”
However, if these glycan tests continue to bear fruit, they offer a tantalizing possibility — a single diagnostic tool that could test for many cancers.
“The approach doesn’t change among cancers,” says Lebrilla. “Ideally, we would develop a single panel that includes markers for several forms of cancer.”