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Protein Biomarker May Lead to Urine-based Test for Childhood Appendicitis

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By Tony Fong

This story originally ran on June 23.

Researchers in Massachusetts have uncovered a novel candidate protein biomarker that could provide the basis for a diagnostic test for appendicitis in children.

If the biomarker pans out in further studies, it would provide clinicians an easy and accurate method for diagnosis of the ailment, which accounts for the most common childhood surgical emergency, according to the researchers.

They describe their work in an article published today in the journal Annals of Emergency Medicine that outlines the discovery of the biomarker, leucine-rich alpha-2-glycoprotein, or LRG.

The biomarker discovery was possible only because of recent advancements in mass spectrometry technology, Hanno Steen, the director of proteomics at Children's Hospital Boston, who directed the proteomics work on the study, told ProteoMonitor last week.

In the study, Steen and his colleagues acknowledge the limitations of their research: the work looked only at patients evaluated in the hospital emergency department setting, and only at children at a mean age of 11. Also, the duration of symptoms in patients for the study was less than one day and the "diagnostic performance of identified markers may be different earlier in the disease course," the authors wrote. In addition, they acknowledge that the biomarker will need further study with patients with underlying renal or urologic disease and those suffering from extreme dehydration.

But Steen said that there is no reason to believe that LRG will not be upregulated in all appendicitis cases.

"In strict terms, at the moment we have a biomarker candidate, we don’t have yet the biomarker but looking at the data so far, we are very optimistic that we can push this further," Steen said.

According to Steen and his co-researchers, because of the heterogeneity of symptoms associated with appendicitis, its resemblance to other conditions — especially ones in which abdominal pain is involved — and a shortage of diagnostic markers, appendectomies are the most common surgical emergency of children.

In the study, the researchers said that though the use of high-resolution computed tomography has led to some improvements in the diagnosis of appendiceal inflammation, the improvements have been "modest." Citing various sources, they said that as much as 30 percent of children who undergo appendectomies do so unnecessarily. On the flip side, between 30 and 45 percent of children diagnosed with appendicitis already have a ruptured appendix, which may prolong and complicate the recovery process.

CT, they add, is being reevaluated because of concerns about risks associated with cancer.

Though appendicitis biomarkers exist — such as peripheral white blood cell count, absolute neutrophil count, and serum C-reactive protein levels — these markers have limited specificity and sensitivity, the researchers said in their paper.

In addition, they said, "Recent attempts to identify new and improved diagnostic markers, such as CD44, interleukin-6, interleukin-8, and 5-hydroxyindole acetate, produced limited improvements compared with the existing [markers], likely as a result of being closely correlated with the existing markers of the general acute-phase response or not specific for the distinct immune mechanisms that characterize acute appendicitis."

For their work, they applied "high-accuracy" mass spectrometry for a differential analysis of individual proteomes in urine samples "and used pattern recognition class prediction and gene expression profiling of diseased appendices to discover candidate diagnostic markers," they wrote. They then evaluated the diagnostic performance of their prospective biomarkers with a blind, prospective study.

LRG a Smoking Gun?

In the discovery phase of their study, they analyzed urine samples from 12 patients, evenly split between those with and without appendicitis, resulting in an initial list of 32 biomarkers, including "known components of the acute phase response" such as alpha-1-acid glycoprotein, plasminogen, and LRG, a biomarker of neutrophil differentiation involved in cell trafficking.

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The researchers added to the list another 25 biomarkers identified by support vector machine learning and through gene-expression studies for a total of 57 candidate biomarkers.

Using immunohistochemistry, they then validated their findings in 67 patients — of which 25 ultimately had proven appendicitis — who had gone to the emergency department at Children's Hospital Boston with possible symptoms of appendicitis: Steen and his co-researchers determined that three proteins — LRG, calgranulin A, or S100-A8, and alpha-1-acid glycoprotein — exhibited "substantial enrichment in the urine of patients with appendicitis," they said in the study.

In particular, LRG was upregulated in patients with appendicitis, at a level almost 500 times more abundant than those without appendicitis. While LRG has been implicated in some cancers, idiopathic normal pressure hydrocephalus, and microbial infections, the AEM study is the first linking the protein with children's appendicitis, which Steen attributed to improvements in mass spectrometry technology. His team used an LTQ Orbitrap from Thermo Fisher Scientific.

"We needed the proteomics because [LRG] hadn't been implicated at all in appendicitis, and if you thought about inflammation markers and inflammatory markers, it wasn't at the top of the list," Steen said. "One would have never followed up on this protein. So in that regard, it was the unbiased proteomic experiment which really convinced us this is a protein that we should follow up."

More research, including further analysis with a larger sample size, and determination of the specificity and sensitivity of LRG as a biomarker for appendicitis, still needs to be performed. In the study, the researchers said that LRG is upregulated in the urine of patients with pyelonephritis, "consistent with its proposed role in local inflammatory processes.

"Consequently, its diagnostic performance of acute appendicitis will likely depend on accurate ability to rule out other local tissue infections, such as pyelonephritis, abscesses, and pelvic inflammatory disease," they said.

But based on their results, "just looking at the receiver-operating characteristics, it showed a much better performance than anything else we have," Steen said.

He added that LRG may have the capability to stratify patients based on the severity of the appendicitis. "The ultimate hope is to have something in hand that can be used in the clinic as a dipstick test or something like that," Steen said.

In the study, the researchers write, "detection of LRG in urine of patients with appendicitis by using Western immunoblotting suggests that widely available clinical diagnostic immunoassays may be devised."

Another avenue that the research team wants to investigate is whether the protein is present in detectable levels in blood. Although urine is the usual test material for patients reporting abdominal pains, in some instances, blood would be a preferable body fluid, Steen said, such as testing for infants, who would need to be catheterized for any urine-based testing.

Another of the candidate biomarkers identified by the researchers, S100-A8, is currently being investigated by AspenBio Pharma as a blood-based appendicitis biomarker.

While the researchers know that LRG is upregulated in patients with inflamed appendices, "we have not managed at this moment to understand the particular molecular mechanisms behind this," and work into this could provide the basis for further study.

"Some type of work in the context of appendicitis has to be done," Steen said.

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