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Stanford Team Uses Mass Cytometry to ID Signatures Predictive of Surgery Patient Recovery Time

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NEW YORK (GenomeWeb) – Stanford University researchers have identified cell signaling signatures that could help predict recovery times for patients undergoing major surgery.

Detailed in a study published this week in Science Translational Medicine, the signatures appeared to explain between 40 percent and 60 percent of the variability in how quickly patients recovered from surgery, Brice Gaudillière, a Stanford researcher and first author on the paper, told ProteoMonitor.

In the future, he added, such signatures could help physicians to determine when patients are best ready to undergo major surgery and possibly provide insights into treatments for patients suffering from slow recovery times.

The researchers performed the study on Fluidigm's CyTOF mass cytometry instrument, using the system to profile immune cells in serial blood samples taken from 32 otherwise healthy patients undergoing hip replacement surgery. In addition to taking molecular measurements on the CyTOF, the researchers tracked patient clinical parameters like fatigue, pain, and functional impairment to determine individual recovery rates.

More than 100 million people undergo major surgery in the US and Europe each year, Gaudillière said.

Yet, he noted, "To date, there is really no diagnostic test that one can perform that can aid physicians to predict how a patient will do after surgery. Patients really don't have the ability to know when they are going to be able to go back to work, when they are going to be back on their feet, or when they will be able to resume their daily activities."

A number of studies have looked at this question, Gaudillière said, but none have provided much in the way of explaining the variance between patient recovery times.

Cytokine levels, for instance, have shown some relationship to patient recovery, accounting for perhaps 10 percent of the observed variance. However, Gaudillière said, with that sort of weak correlation, "it's difficult to make the leap and say this is clinically relevant."

Many of these studies – the Stanford STM work included – have focused on the immune system, the notion being that surgery is a major trauma and as such produces a significant immune response. The key difference in the Stanford effort, suggested university researcher Gary Nolan, senior author on the paper, is the breadth and depth of analysis enabled by the CyTOF instrument.

Originally developed by DVS Sciences, which was acquired this year by Fluidigm for $207.5 million, the CyTOF combines capabilities of flow cytometry and atomic mass spectrometry, allowing it to measure large numbers of proteins in single cells with high throughput.

Atomic mass spectrometry detects proteins using antibodies linked to stable isotopes of elements, which can then be read with high resolution via time-of-flight mass spectrometry. The platform is able to simultaneously quantify as many as 100 protein biomarkers in individual cells at a rate of roughly 1,000 cells per second.

This, Nolan told ProteoMonitor, allowed him and his colleagues to characterize the immune cells in surgery patient blood much more thoroughly than was possible in past efforts. Nolan was an early adopter of the instrument and has played a significant part in technology development for the device.

"The value of the CyTOF is its ability to do dozens of markers per cell, which lets you look at combinations of markers and intracellular signaling proteins that just can't be read out by conventional flow cytometry," he said.

The Stanford team looked at 21 surface markers, which let them identify and quantify specific immune cell types, and 10 protein phosphorylation markers, which allowed them to look at the activation states of signaling pathways within these cells.

It was this latter bit of information that ultimately proved useful, Nolan noted. While the frequency of different cell types didn't differ significantly between patients regardless of their recovery time, the researchers found that patients who were quick to recover had different patterns of pathway activation in a particular type of cell, CD14+ monocytes. In particular, subsets of these cells exhibited changes in STAT3, CREB, and NF-Кβ activation states that correlated with patient recovery.

"In [previous] studies all [researchers] were looking for was the presence or absence of different cell types based on the surface markers that people commonly use," Nolan said. "Whereas we not only had all of the cell types and many more than people were able to look at previously, but we were also able to look intracellularly at key signaling proteins."

The large number of parameters the CyTOF was able to look at also allowed Nolan and his colleagues to delve deeper into their samples, more finely slicing the different cell categories. Ultimately, he noted, the signature they identified for distinguishing between fast and slow recovering patients was based on information from around only 1 percent of the total cells in the sample.

The CD14+ monocytes that the researchers identified as key to predicting patient recovery times are involved in regulating the body's initial immune response.

"The idea behind these cells is that the moment you activate an inflammatory response you also are preparing to suppress the response," Nolan said. "If you didn't have those cells you would get runaway inflammation. So what we think is going on is that the patients who go for a long time in the hospital [after surgery] are those who are not coordinating the shutdown response appropriately."

"Some people feel like they are ready to roll between five and seven days [after hip replacement surgery]," Gaudillière said. "Others it takes weeks, and even after six weeks they are still not fully recovered in terms of fatigue and pain and function."

The Stanford team hopes to use their findings to predict which patients will behave in which way. The assay presented in the STM paper used blood taken 24 hours after surgery, but, Gaudillière said, the researchers are now trying to develop an assay for use before surgery. Such a test could potentially allow clinicians to better tailor the surgery for patients likely to require longer recovery times or even let clinicians determine whether or not a particular patient is "immunologically fit to go to surgery," he said, adding that they have started a prospective clinical trial involving around 100 patients to look at effectiveness of a pre-surgery assay.

Additionally, the research could provide insight into the biology of the healing process and treatments that might help speed it along. For instance, Gaudillière and his colleagues are currently running a clinical trial to see if arginine – which past studies have suggested could aid in surgery recovery – can modify CD14+HLA-DR(low) monocytes in patients, whose activity highly correlated with patients recovery.

Further down the line, he said, is the possibility of using drugs targeting the transcription factors -- STAT3, CREB, and NF-Кβ – whose activation in CD14+HLA-DR(low) monocytes were strongly associated with how quickly patients recovered from surgery. Some inhibitors to these proteins (like JAK/STAT inhibitors) are already FDA approved and currently used in management of transplant patients, he noted.

"Of course, targeting these molecular mechanisms to improve patients surgical recovery is a long term project, but a promising research avenue with important clinical implications," he said.

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