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Theranostics Health Launching Alternative to Formalin for Preserving Phosphoproteomic Tissue Samples


By Adam Bonislawski

Theranostics Health has licensed a new fixative developed by researchers at George Mason University that could significantly improve the quality of tissue samples available to proteomic researchers.

In particular, the fixative, which the company has named TheraLin and plans to bring to market sometime this fall, could improve the preservation of protein phosphorylation and immunohistochemical activity compared to traditional formalin fixation, as well as lower costs compared to snap-freezing methods, Emanuel Petricoin, co-director of GMU's Center for Applied Proteomics and Molecular Medicine and one of the inventors of the reagent, told ProteoMonitor.

While discussions of proteomics' limitations often focus on questions of analytical capabilities – mass spec sensitivity or antibody specificity, for instance – effective sample collection is also a significant problem for the field, said Petricoin, who is a founder of Theranostics and the chair of its scientific advisory board. This is especially true in the case of protein phosphorylation, which is a highly labile post-translational modification.

Phosphorylation has emerged as a prime area of interest within proteomics, particularly in the pharmaceutical and clinical arenas where researchers are studying phosphoproteins in hopes of developing and better targeting kinase inhibitors for the treatment of a variety of diseases, including many cancers. The lack of a good method for preserving tissue samples intended for phosphoproteomic research, however, has been a significant impediment to this work.

Formalin fixation, which is the most commonly used method for preserving tissue biopsies, works slowly, taking hours to penetrate and fix a sample. Additionally, the process can reduce the immunohistochemical activity of the proteins in the tissue, and yields of proteins extracted from sample preserved in this manner are typically poor. Snap-freezing, on the other hand, works quickly, but the equipment needed to process and store these samples is expensive and unavailable in many clinical environments.

"It's not just that the tissue is losing phosphorylation," Petricoin said. "It's that the tissue is alive when you take it out [of the patient], and you have changes in phosphorylation going on as the tissue reacts. If you're going to give somebody an AKT inhibitor in a clinical trial because they have high AKT pathway activation, and it turns out that it was high because the tissue sat out too long, that's not a good thing."

Petricoin and Theranostics have a particular interest in solving this problem given that much of their work focuses on protein phosphorylation. Petricoin, with his fellow CAPMM co-director and Theranostics co-founder Lance Liotta – also one of the inventors of the new fixative – is currently developing a database detailing phosphorylation levels in a variety of protein-signaling pathways linked to common cancers (PM 08/13/2010).

Theranostics, which the pair launched in 2006 to commercialize their laser-microdissection and reverse-phase protein microarray technologies, specializes in developing phosphoprotein profiles to aid in drug development and targeting.

The idea for the fixative, Petricoin said, came out of a discussion several years ago with Richard Gaynor, vice president of oncology at Eli Lilly, who told him that although phosphoproteins could be hugely important as biomarkers, pharma researchers would be hesitant to embrace them until the problem of stabilizing and preserving phosphorylation in patient samples was solved.

"Especially in big Phase III clinical trials, there's not always going to be the ability to snap freeze tissue," Petricoin said. "At some clinical site in India or out in the middle of the US somewhere away from a big medical center, tissue is going to be dropped into formalin and that's it. So you're going to need to come up with some other solution if you think phosphoproteins are going to be used in the clinic."

Indeed, according to Amanda Paulovich, a researcher at the Fred Hutchinson Cancer Research Center, sample collection is a major concern with regard to phosphorylation work planned for the second stage of the National Cancer Institute's Clinical Proteomic Technologies for Cancer initiative, which kicked off last week (PM 08/26/2011). Paulovich is leading one of CPCT's proteome characterization centers, as well as chairing the network's steering committee for the first year.

The CPCT researchers "are extraordinarily concerned about" sample collection, she said, noting that the issue was "given a lot of airtime" during the initiative's kick-off meeting last week. A major concern, Paulovich told ProteoMonitor, is that tissue samples the group has obtained from the NCI-funded Cancer Genome Atlas may not be appropriate for phosphoprotein analysis given that they were originally collected for genomic, not phosphoproteomic, research.

"Those samples were collected very rigorously with great quality control with genomic analysis in mind, but of course they had no idea that downstream [the samples] would be co-opted for use in proteomics," she said. "So they weren't really collected under ideal conditions for the analysis by proteomics of things like post-translational modifications."

"There are different considerations when you're talking about molecules as labile as the phosphoproteome that respond within seconds to cells being stimulated or perturbed," she added.

Given these issues, it's possible that the CPTC researchers will forgo phosphoproteomic work on the TCGA samples and instead do "prospective sample collection done under a protocol that we know works" for preserving phosphorylation, Paulovich said. "The plan is to have a quality control process in place for which any PTM profiling has to pass in order to qualify for network-wide analysis, whether it be the TCGA [tissue] or other samples."

In a study published in PLoS One in August, the GMU researchers demonstrated that TheraLin preserved protein phosphorylation at a level comparable to snap-freezing, while maintaining the diagnostic immunohistochemical and histomorphological detail of formalin fixation.

This later capability is important, Petricoin noted, because, even if a fixative were to offer improved phosphoprotein stability, it likely wouldn't find wide use if it didn't also preserve the tissue's histopathology and immunohistochemistry.

"Having something that is just good as a phosphoprotein stabilizer would never be used in a molecular pathology or personalized medicine space because the same piece of tissue has to give you formalin-like histology and it has to give you the ability to do formalin-like immunohistochemistry," he said. For example, "every breast cancer is immunohistochemically stained for [estrogen receptor], [progesterone receptor], and HER2, so if your fixative doesn't provide identical histology and formalin-like staining for those three proteins, it doesn't matter what else it's good for, it will never be used."

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Even if the results from the PLoS One study hold, though, convincing pathologists to switch to the new fixative could prove challenging, Jon Burrows, vice president of R&D at Expression Pathology, which specializes in applying selected-reaction monitoring mass spec to formalin-fixed, paraffin-embedded tissue samples, told ProteoMonitor.

"It's a good idea, but, the thing about it is, you have to change clinical practice to do it," he said. "And that's just a huge hurdle. I actually like their idea, but when you talk to doctors, they say, 'Well, we'll do it to see if looking at the phosphorylations is that important, if it gives us a leg up, but we don't seeing it as being a mainstream thing.'"

Petricoin acknowledged this, saying that "what we're trying to do is position this fixative not so much as a formalin substitute – because that would require a huge amount of work by people like the College of American Pathologists to do formal crossover studies. We're trying to position it as the fixative for personalized medicine and molecular profiling where molecular pathologists and oncologists and the like would order it to be used [for tissues] that are going to be used for molecular profiling."

According to Ron Hencin, Theranostics' vice president of business development, several of the firm's clients have begun using TheraLin on a trial basis. Theranostics is also talking to a number of clinical contract research organizations, which, he told ProteoMonitor, could help raise awareness of the fixative among oncologists.

TheraLin is also slated to be included after its commercial release in the NCI's Cancer Human Biobank Program led by Office of Biorepositories and Biospecimen Research director Carolyn Compton, Hencin said. He noted that "if we could get it adopted and in use for that program, I think it would go a long way toward providing that validation the pathologists would look for."

Ultimately, Theranostics plans to partner with a larger vendor like "a Becton Dickinson" to market and distribute the product, he said. "We don't feel that we really want to be bulk distributing fixative. That's not really our main line of business here."

Hencin added that Theranostics is also working on a version of TheraLin for use with blood, noting that such a reagent could be useful "in the flow cytometry arena where there is no good solution for [preserving] phosphoproteins and some surface markers."

Efforts are also afoot to improve the utility of formalin fixing for proteomics research. Biotech firm Pressure Biosciences, for instance, has applied for a Phase I SBIR grant in support of its efforts to develop a pressure-based platform to speed up formalin fixing of clinical samples. According to PBI CEO Richard Schumacher, using the platform the company thus far has achieved a five- to seven-fold increase in the speed of sample fixation, which could improve preservation of proteins, including those with PTMs like phosphorylation.

Have topics you'd like to see covered in ProteoMonitor? Contact the editor at abonislawski [at] genomeweb [.] com.