The delineation of UC and CC has to date been based on the use of a combination of potentially clinical, endoscopic, histologic, and/or radiographic assessments that are utilized to guide both medical and surgical therapy. Despite advances in our understanding of the genetic,22,23
factors that may contribute to the pathogenesis of IBD, there is no pathognomonic indicator sensitive enough to accurately and consistently discriminate CC from UC. MALDI-MS has proven to be an effective means to assess the protein microenvironment of tissue layers.15–17
MALDI-MS methodology used in a histology-directed manner permits us to target highly specific areas in colonic tissue for analysis without the need for extensive sample preparation, such as that needed for laser capture microdissection.15
This targeting allowed us to specifically assess the colonic mucosa and submucosa with MALDI-MS since the structural integrity of the tissue was maintained (as opposed to proteomic assessments using full thickness or morselated colonic tissue). In addition, the histology-directed approach permitted specific pathological assessments of inflamed and uninflamed areas within the mucosal and submucosal layers. Our study is the first report using histology-directed MALDI-MS to compare proteomic patterns in the colonic tissue layers in order to distinguish differences between the two inflammatory colitides.
Histology-directed mass spectral protein analysis was able to identify colonic protein signatures that statistically distinguished inflammation in the submucosal layer of CC with a classification (LNOCV and LOOCV) accuracy of 86 percent, and to a lesser extent, in the UC specimens with an accuracy of 75 percent. This lower accuracy may be due to a lesser degree of inflammation in UC versus CC, different types of disease activities or inflammation, or due to the low sample numbers in this study. Another possibility could be because of the statistical high dimensional variable selection based on molecular features of disease on cross-validation results.26
Further validation studies are ongoing with greater numbers of samples to better assess the ability of MALDI-MS to distinguish inflammation in the submucosa of UC as well as CC specimens. The addition of normal colonic submucosa as controls in our comparisons may better assess proteomic differences to truly see if MALDI-MS can distinguish inflamed and uninflamed submucosa.
When using histology-directed MALDI-MS to compare proteomic profiles of CC and UC inflamed and uninflamed submucosal layers, five differentially expressed m/z values were identified that could distinguish the colitides. These proteomic peaks were noted to show greater intensity in the CC submucosa, perhaps indicative of the greater degree or different type of inflammation in the tissues underlying the mucosa. It is possible that these differing peaks may represent candidate biomarkers that could be utilized to delineate the inflammatory colitides. This pilot study is the first of its kind to utilize tissue assessment with MALDI-MS to delineate CC and UC.
There has been great interest in trying to identify protein biomarkers that might delineate the inflammatory colitides. These studies have been variably successful using serum,27,28
biomarkers as prognostic indicators or in assessing whether the IBD is in a quiescent or active state. However, these biomarkers have not been shown to distinguish UC from CC, but rather as prognostic for the known diagnosed disease as being quiescent or active.27–34
Recently, Meuwis, et al.35
reported the first proteomic study on the sera of IBD patients using surface-enhanced laser desorption/ionization time-of-flight mass spectrometry technology, and they identified four biomarkers (PF4, MRP8, FIBA and Hpα2) of interest. These biomarkers had been identified previously in the setting of IBD36,37
and only serve to confirm the presence of IBD rather than differentiating the disease phenotypes. The fact that these biomarkers were selected through a robust statistical process among a large range of potential serological markers highlights their potential importance in both IBD pathophysiology and diagnostic confirmation. The potential biomarkers they identified are small proteins or fragments of proteins (or small protein isoforms potentially with variable posttranslational modifications) that would not necessarily be easily detected by strategies other than proteomic analysis and mass spectrometry. Ansari, et al.,38
in another recent study showed significantly higher expression of serological RANTES (Regulated on Activation, Normal T cell Expressed and Secreted) in UC patients rather than CC patients. It is unclear, as of yet, whether the proteins from these reported studies correspond to the proteomic peaks that we are currently in the process of identifying in our study using MALDI-MS.
There are a number of limitations in our study. The major one is the small sample size of 51 patients (n=24 CC and n=27 UC) which renders the estimation of statistical significance of the differences between groups approximate. It also limits the evaluation of classification to LNOCV performance without the independent validation of the test set. While the data indicate statistically significant differences of m/z peaks used in classification, studies of larger numbers of patients are needed to validate these results on the independent sample cohorts.
The fact that inflammation in the UC submucosa in our study showed features with average p-values in the range from 1.5 to 5.5×10−3
(and accuracy of 75 percent) by MALDI-MS may be due to (apart from other reasons such as inadequate sample size) the extent of inflammation being limited to the mucosa38
although the submucosa is often also inflamed10–12
as we also noted in our studies6,7
and have confirmed that there is some degree of an inflammatory and immune response taking place in this layer that can be studied by mass spectrometry.6–9
Our study is also limited by how we determined the underlying diagnosis of UC and CC by the current gold standard of histology using H & E. As noted in the introduction, this method of diagnosis (in combination with other clinical and diagnostic techniques) may be incorrect in up to 15 percent of cases making CC appear to be UC. Even if up to 15 percent of the UC samples that we examined were in fact CC, this would actually bias the data toward the null hypothesis when comparing the proteomic patterns between the inflamed and uninflamed submucosa of the two colitides, but statistically significant differentiating peaks were still identified in our study. Another limitation is the lack of a normal colonic submucosal control for comparison. While we wanted to focus on the proteomic differences between the colitides (which did not require a normal control), our further validations of MALDI-MS as an effective technique for identifying tissue inflammation will include normal controls to better determine its effectiveness.
The distinctive m
values that we identified showed statistical differences between CC and UC only when profiling the submucosal layers. Protein signatures obtained from inflamed mucosa showed discrimination between UC and CC, but the accuracy of classification estimated by LNOCV was rather low. However, there is a possibility that better results can be achieved on a larger sample set. This may limit the clinical usefulness of our results when assessing endoscopic biopsies of the colon given that these most abundantly include the mucosa. Submucosal layer biopsies, however, although limited, may as well be obtained endoscopically.39–41
The need to evaluate submucosa to differentiate UC and CC with MALDI-MS is therefore a potential limitation, except for assessment of colectomy specimens, given that submucosal tissue is abundantly available after surgery.
Our study provides potential opportunities for future analyses. While the pattern of proteomic expression and/or the protein localization patterns may hold diagnostic potential, individual proteins may not conclusively be identified by molecular weight (m/z) alone. Analyses are underway to identify the signals to see if they represent discriminative proteins. Identified proteins may be used in studies on endoscopic biopsies or, if confirmed, as serum biomarkers for discrimination between CC and UC (and even as differentiators of indeterminate colitis). Protein identification and validation may provide differentiating biomarkers that would not only improve diagnostic accuracy but may aid in more appropriate, personalized treatment of UC and CC patients and may also provide the basis for future studies on the underlying pathophysiology of IBD.
In conclusion, we found that MALDI MS tissue profiling as described significantly distinguished the inflammatory colitides. Significant discriminatory m/z peaks were identified in these pilot proteomic analyses of both inflamed and uninflamed colonic submucosa from UC and CC. Histology-directed MALDI MS profiling allows for the analysis of high enriched cell types and has the power to predict protein differentiation that can allow inflammatory colitis delineation via submucosal analyses. The methodology revealed 8 m/z peaks of interest, and of these, 5 peaks were individually considered as “good classifiers”. Further analyses and protein identification may provide differentiating biomarkers that would not only improve diagnostic accuracy but may aid in the personalized treatment of UC and CC.