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Louisville Team and Spinout LBIdx Exploring Protein Denaturation Profiles as Disease Markers

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University of Louisville researchers have identified changes in the thermal behavior of plasma proteins that could prove useful in the detection of cervical cancer.

Detailed in a paper published last week in PLOS One, the study used analysis of plasma protein thermograms — protein denaturation profiles generated using differential scanning calorimetry. These profiles, the U of L researchers and others have found, are often altered under disease conditions, making them potentially useful as diagnostic biomarkers.

Several of the study authors are also founders of Louisville Bioscience (LBIdx), a diagnostics firm spun out of U of L in 2008 to commercialize the plasma thermogram technology. LBIdx is currently in the midst of a Series A funding round targeting $1.5 million to support the 2015 launch of its first product, a plasma thermogram-based test for monitoring melanoma patients, CEO Mark Wisniewski told ProteoMonitor.

In the PLOS One study, the U of L team examined plasma samples from 32 women with the precancerous condition cervical intraepithelial neoplasia (CIN), 35 with invasive cervical cancer (IC), and 4 health controls. Analyzing plasma thermograms from each sample, the researchers were able to distinguish CIN and IC from healthy controls as well as early IC from advanced IC.

Though still in the relatively early stages of development, plasma thermogram-based analysis has shown promise in detecting a variety of conditions ranging from cancers to autoimmune diseases. A significant potential advantage of the technique is its simplicity and robustness, U of L researcher and LBIdx co-founder Nichola Garbett – an author on the PLOS One study – told ProteoMonitor.

While most protein biomarker work has focused on measurement of low-abundance markers, which presents a host of analytical challenges, plasma thermogram tests typically look at high-abundance proteins like albumin and immunoglobulin G.

"It's very straightforward," Garbett said. "You just take plasma or serum, put it in the [differential scanning calorimeter] and collect the profile."

The simplicity of the workflow also makes for low experimental variability, she said, adding that the technique also appears to be quite robust in terms of variability introduced by sample collection and storage methods.

The researchers discovered the phenomenon serendipitously, Garbett said. "We were looking at plasma proteins and plasma changes in general, and we began to see that we would get these [denaturation] profile changes [in diseased samples] that were different from healthy controls."

"Then we began to explore various diseases and we saw these characteristic changes, and then a number of other groups have explored it since in more diseases and conditions and also seen changes," she said.

Despite its apparent promise, the method does face one significant challenge, Garbett noted – researchers aren't entirely sure what is causing the change in the plasma thermograms.

"That's the big question right now," she said. "And we haven't really shown definitively what is causing the changes."

One theory is that disease conditions could lead to changes in post-translational modifications on the high-abundance proteins the researchers analyze, leading to shifts in their denaturation profiles. Other possibilities, she said, include interactions with other proteins, peptides, or lipids.

While it might be possible to develop pattern-based diagnostics without identifying the precise analytes responsible for these patterns – as in the case of Biodesix's MALDI-based proteomic lung cancer test Veristrat – ideally, Garbett said, they would identify the underlying analytes.

"At least for me – and for some reviewers that we've had – it adds more value if you can understand the mechanism of the changes," she said.

Another challenge of a pattern-based diagnostic is developing patient classification methods straightforward enough for clinical use, Garbett noted.

"It's not like an ELISA where you just get a number and you compare the numbers," she said. "There's quite a high information content [in the profiles]."

"We have methods where you can compare the shape and amplitude of these distributions and come to some strategy for how to classify a test sample, but it's not immediately straightforward, and so that is something we need to develop further for easy clinical application," she added.

In the PLOS One paper, Garbett and her colleagues used mass spec to look for molecules that might be causing the profile shifts they observed in the CIN and IC samples. Analyzing the low molecular weight fraction of patient plasma and urine samples using MALDI MS on an Applied Biosystems 4700 Proteomics Analyzer, they identified a number of peptides whose abundance correlated with the extent of cervical disease.

According to Garbett, the researchers plan to continue coupling the plasma thermogram method to mass spec analysis as they move on to further analysis of clinical samples. In particular, she said she is interested in applying both techniques to longitudinal studies looking at the effect of therapies and disease recurrence.

"That is our principal route right now, [longitudinally] monitoring patients to understand [plasma thermogram] changes with respect to their clinical status and coupling that to mass spec analysis," she said.

Currently, the primary clinical focus for both the U of L researchers and LBIdx is melanoma. The LBIdx test scheduled for launch next year is intended as an auxiliary to imaging techniques like PET and CT scans currently used for monitoring melanoma patients, Wisniewski said. Because it would not expose subjects to radiation, such a test could allow for more frequent monitoring of patients either under treatment or being watched for recurrence.

The company recently completed a roughly 100-subject phase I trial looking at plasma thermograms as indicators of remission in melanoma patients and are now planning a 30- to 50-patient phase II trial investigating the test's usefulness for monitoring response to therapy in late-stage patients.

According to Wisniewski, the company plans to launch the test as a CLIA test and is in negotiations to obtain access to a CLIA lab.

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