Researchers at the Universities of Florida and Michigan have developed a technique to profile glycoproteins in urine with the goal of using a panel of the biomarkers to develop a test for bladder cancer.
Using their technique, which combines concanavalin A lectin affinity chromatography with liquid chromatography-mass spectrometry, the researchers were able to use far fewer urine samples than in previous efforts to find bladder-cancer related proteins.
And while results are very preliminary, they were able to find five proteins that appear to be specific to bladder cancer. The study appears in the July 6 edition of the Journal of Proteome Research.
According to the National Cancer Institute, this year 67,160 new cases of bladder cancer will be diagnosed in the US with 13,750 deaths, making it the sixth most-common cancer in the country. An estimated $2.9 billion is spent annually for treatment.
Various methods are used to detect the disease including physical exams, injecting a dye into a patient where it collects in the bladder making it visible on X-rays, and inserting a cytoscope through the urethra into the bladder to examine the lining.
The most common non-invasive diagnostic technique is voided urine cytology where a urine sample is examined under a microscope for cancer cells. However, VUC has a specificity of about 25 percent to 40 percent, according to the Florida researchers, rendering them ineffective for detecting low-grade and low-stage tumors.
“When they find very odd looking cells, then it’s quite accurate, but they miss an unacceptable percentage of cases where they think there’s nothing wrong but there was,” Steve Goodison, an associate professor in the department of surgery at the University of Florida and the senior author of a report detailing the work by the research team, told ProteoMonitor this week.
Indeed, the American Cancer Society says on its website that while low-grade bladder cancer is rarely life-threatening, “a more sensitive test for low-grade cancers would be an important advance.”
Two protein-based tests approved by the US Food and Drug Administration — the bladder tumor antigen test and the nuclear matrix protein 22 test — have the opposite problem of the VUC test.
“They have an improvement on sensitivity. Tthe problem is they all suffer from a lack of specificity,” resulting in a high rate of false positives, said Michael O’Donnell, professor of urologic oncology at the University of Iowa, College of Medicine.
The BTA and NMP22 test detects for cancer based on the presence of one protein. What the University of Florida researchers are doing is looking for panels of biomarkers.
That approach, O’Donnell said, will cut down on the rate of false positives.
“If you can develop a panel that is high-throughput … and cost-effective, that would be a great approach,” he said.
Goodison and his team are still years away from developing their test, but they say in their study that their “combinatorial approach provides high sensitivity and with relatively moderate labor demands should facilitate the identification of potential biomarkers of disease from body fluids.”
Less Urine, Tests Better
The approach developed by Goodison and his colleagues involves profiling N-linked glycoproteins in urine. The team chose urine as the basis for its test for several reasons. One is that in comparison to other fluids, especially blood, urine is much simpler with a lower dynamic range.
Also, because the bladder is lined with cells that shed, much like skin, “you get a lot of cells shedding into the urine naturally, and if you’ve got a cancer tumor inside the bladder, you get a lot more cells and you get a lot more proteins.” Goodison said. “So it’s relatively easy to detect, if only we can find the proteins that are specific to bladder tumors over infection or other lesions.”
Urine samples were collected from the University of Florida urology clinic and Shands at the University of Florida hospital. A total of 10 samples were collected, split evenly between patients with confirmed cases of bladder cancer and controls.
After samples were collected, cells and debris were removed by centrifugation at 5,000 grams for 10 minutes at room temperature. The supernatant was then frozen at minus-80 degrees Celsius until analysis. For protein precipitation, four times the sample volumes of acetone at minus-20 degrees Celsius were added to the urine samples. The supernatant was removed after the sample was left at 20 degrees Celsius for one hour followed by centrifugation at 12,000 grams for 15 minutes at 4 degrees Celsius.
“If you can develop a panel that is high-throughput … and cost-effective, that would be a great approach.”
The Bradford protein assay, produced by Bio-Rad Laboratories, was used to determine protein amounts in the sample concentrates.
Agarose-bound concanavalin A lectin affinity chromatography was performed to enrich N-linked glycoproteins, which were then tryptically digested. A Paradigm MG4 micropump from Michrom Bioresources was used for chromatic separation of peptide mixtures. After the peptides were resolved, they were then analyzed on a Thermo Finnegan LTQ linear ion trap mass spectrometer with a nano-LC-ESI source.
MS/MS spectra were analyzed by the TurboSequest module of Thermo Scientific’s Bioworks software by Thermo Scientific. Peptide fragment lists were generated and submitted to SwissProt database searching. Protein identification was accepted only if they were positively identified in at least two MS/MS analyses, the authors said.
In total, Goodison’s team identified 186 proteins, including five that were present only in patients with cancer.
The results still need to be validated, but one of the key features coming out of the research, Goodison said, is that only small amounts of urine — 15 milliliters to 20 milliliters — were needed for each sample analysis.
“It’s really a technical advance where we have got the techniques down to such a point where we can identify almost all of this subset of proteins that comes from 10 samples with very small amounts of urine,” he said. “Previously people have profiled urine proteins, but they’ve used liters of a sample, and it’s taken them many months to do it.
“Now with improved techniques, which keep improving all the time, we can do this relatively quickly and with just a small starting sample of about 15, 20 milliliters of urine which is something you typically get from a patient instead of having to do it with liters of urine,” he said.
Because he and his colleagues are only in the first of three phases and the sample size was so small, Goodison was careful to stress that the findings are still very preliminary. Further profiling of proteins needs to be done, followed by validation, leading up to assay development. The ultimate goal is to develop either an ELISA-based diagnostic or a dipstick test.
“If you could test the urine with a proteomic-based test, that would be great for the patient. And it would be good for the health care system as well,” Goodison said.