While formalin-fixed, paraffin-embedded tissue is widely used in light microscopy research, its usefulness in proteomics work remains an open question.
But Expression Pathology, which develops technology that enables researchers to use mass specs on FFPE samples, is aiming to change that. Last month, the Gaithersburg, Md.-based firm won a $148,000 in Phase I Small Business Innovation Research grant from the National Cancer Institute to use its technology to identify protein biomarkers in primary breast cancer tissue that differentially express during early-stage metastasis.
Since its founding five years ago, Expression Pathology has focused on developing the technology to allow for proteomic analyses of FFPE tissue. The company now is transitioning from an early-stage development business to one that markets and sells consumables and eventually develops and sells diagnostic tests.
“The whole idea has been to be able to finally exploit formalin-fixed tissue not only because there are these archived collections with clinical outcome data … but because that’s the tissue that goes into clinical labs every day,” said Casey Eitner, president and CEO of Expression Pathology.
For proteomics work with FFPE tissue, immunohistochemistry has been the only technology available, but the method is “qualitative and it’s subjective [and] frequently cannot work with certain proteins,” he said.
The issue has been that with FFPE tissue, the samples create crosslinks that make proteins insoluble and therefore unsuitable for routine biochemical analysis. Expression Pathology, however, has developed two technologies to overcome these hurdles: Liquid Tissue MS protocol and the Director Laser Microdissection Slides.
The Liquid Tissue protocol is a method for solubilizing and capturing the total protein content of microdissected FFPE tissue. Starting with 30,000 cells, “which you can typically harvest from a single 10-micron section” of a tissue block, a researcher can pick off the cells needed, Eitner said. According to Expression Pathology’s website, the technology can identify more than 2,000 proteins from FFPE samples using LC-MS/MS analyses.
The Director slides use a proprietary technology to replace plastic films and adhesives for direct non-contact microdissection. They work with existing Leica and Zeiss microdissectors, “improving their speed and [automation] and precision,” Eitner said.
By using a combination of heat buffers and enzymatic digestion — “which until we came along had never been demonstrated or applied to solubilized FFPE tissue,” Eitner said — Expression Pathology’s technology works to undo the crosslinking that occurs in the tissue.
“The whole idea has been to be able to finally exploit formalin-fixed tissue not only because there are these archived collections with clinical outcome data … but because that’s the tissue that goes into clinical labs every day.”
In addition, the company’s method eliminates separation steps, meaning “there are no compartments of proteins that are discarded or lost in the process,” he said.
The SBIR award is focused on discovery work: Researchers will be examining early-stage tumors that clearly have metastasized, those that clearly haven’t, and those whose evaluations are not as clear.
According to Eitner, previous work on the company’s platforms has proven their utility and efficacy. Doing global proteomics profiling, the company was able to identify biomarkers associated with metastasis on a small-scale NCI-sponsored project done a few years ago.
And at the annual conferences of the American Association for Cancer Research and the American Society of Mass Spectrometry, Toshihide Nishimura at the Tokyo Medical University presented a study comparing non-small cell lung cancers in which he and colleagues identified differentially expressed candidate biomarkers.
Further, using aliquots from Liquid Tissue preps, Nishimura’s team performed multiple-reactive monitoring on mass spectrometry and verified and validated certain biomarkers correlated with metastasis, including one that was down-expressed in the primary tumors that had metastasized and appear to be “highly predictive of poor prognosis,” Eitner said.
The company chose to focus the SBIR grant on breast cancer because of its prevalence. Despite a drop in the number of new cases, breast cancer remains the most diagnosed cancer in the US, according to the National Cancer Institute. This year more than 182,000 American women will be diagnosed with the disease. The disease will kill more than 40,000 women this year.
While there are commercially available gene-expression assays that help distinguish early-stage breast cancers, “we believe that at the end of the day, the researcher and the clinician is going to prefer to understand that molecular pathology on the protein level because it’s the proteins that are the actual actors,” Eitner said.
Both the Liquid Tissue and Director technologies will be used to conduct the research. The end goal is to create a laboratory-based diagnostic test.
Though few proteomics researchers use FFPE tissue in their work, Expression Pathology’s business model has always been based on FFPE technology. The strategy — building a company around rarely used technology — may seem counter-intuitive, even foolhardy, but the two founders of Expression Pathology, David Krizman and Marlene Darfler, both had extensive experience with tissue work, especially FFPE, and saw the potential of the tissue in proteomics, according to Eitner.
“We kind of look at ourselves like a replay of gene expression,” Eitner said. “If you go back and look at gene expression 10 years ago, people were jumping all over the opportunity to identify differentially expressing genes initially in frozen tissue and more recently in FFPE tissue. And certainly the quality of that data dramatically improved when laser microdissection came along.”
Both the Liquid Tissue and Director technologies are commercially available, and Expression Technology has a service business using them to do proteomic profiling and MRM and single-reaction monitoring protein quantitation projects using FFPE tissue for its pharma and biotech clients.
Eitner declined to identify those clients, but said that the company also has relationships with mass-spec contract research organizations, including Monarch Life Sciences and NextGen Sciences.
This week, NextGen and Express Pathology announced a co-marketing and joint service offering agreement covering the former’s mass specs and the latter’s Director and Liquid Tissue technologies, and both parties’ bioinformatics. The deal is worldwide and non-exclusive. They declined to disclose financial terms.