Proteomic Analysis of Plasma Exosome-Associated Proteins Reveals That Differences In Kappa:Lambda Ratios Predict Severe Acute Graft-Versus-Host Disease Early After Allogeneic Hematopoietic Stem Cell Transplantation.
Authors: Alge Joseph L , Janech Michael , Schwacke John , Arthur John , Costa Luciano J 
Published: October 2019

Abstract
Abstract 1278

Background:
One of the chief obstacles to improving the outcomes of patients with acute Graft-Versus-Host disease (aGVHD) is accurate and rapid diagnosis. Currently it is diagnosed clinically, once the symptoms are fully manifested and most of the implicated biological processes are established. A biomarker that could predict disease during the asymptomatic period would allow for earlier intensification of immunosuppressant therapy, which could improve patient outcomes. Exosomes, externalized membrane bound nanovesicles that are derived from the multivesicular body, are an attractive target for aGVHD biomarker discovery. They contain a variety of cellular components and are easily obtained from bodily fluids, including plasma. Furthermore, the cell types implicated in the pathophysiology of aGVHD are known to produce exosomes with potent immunomodulatory effects. We utilized a proteomic approach to identify biomarker candidates among exosome-associated proteins utilizing patient samples obtained early after allogeneic hematopoietic stem cell transplantation (allo-HSCT).

Methods:
We utilized a differential centrifugation protocol to isolate exosomes from cryopreserved plasma and confirmed the presence of exosomes in the final pellet both by Western blotting of known exosome markers and by electron microscopy. We then used this protocol to isolate exosome-enriched pellets from 5ml plasma samples obtained on posttransplantation day 7 from 3 patients (cases) who later developed severe aGVHD (grade 3 or 4) and 4 patients did not develop aGVHD of any grade (controls). We performed proteomic analysis on pellets using liquid chromatography-tandem mass spectrometry and iTRAQ labeling, which allowed us to compare the relative quantities of identified proteins between cases and controls.

Results:
We made 33 protein identifications. iTRAQ analysis comparing the relative abundances of the identified proteins in cases and controls did not demonstrate any statistically significant differences between the two groups. This is likely due to the small sample size of our dataset. Despite this limitation, our data did show trends toward differences in the relative abundances of some proteins. Specifically, there was a trend toward increased IgG3 constant region in cases (median case:control ratio= 1.36; credible interval of case:control ratio= 0.857, 2.19). Additionally, there were trends toward increased lambda light chain constant region in cases (median case:control ratio= 1.35; credible interval of case:control ratio= 0.844, 2.07) and decreased kappa light chain constant region in cases (median case:control ratio=0.847; credible interval of case:control ratio=.0586, 1.21). We performed further statistical analysis to compare the kappa:lambda ratios in cases compared controls. Briefly, we calculated a ratio of treatment effects by dividing the case kappa:lambda ratio by the control kappa:lambda ratio. A value of 1 would indicate that the ratios do not differ between cases and controls. We observed a median value of 0.63, with a 95% CI of 0.377, 1.027 (see Figure 1 for a histogram of the posterior distribution of kappa:lambda effect ratio). Comparing the median treatment effect of the kappa:lambda ratio to a null distribution which we generated by bootstrap sampling 5,000 pairs of proteins that were identified in our iTraq analysis, we calculated a p-value of .011.

Conclusion:
These results indicate that there are early differences in B cell activity between patients who go on to develop severe aGVHD and those who do not. Monitoring allo-HSCT patients for markers of B cell activation and expansion could be clinically useful with regard to early diagnosis of aGVHD. Further studies will be conducted to test the hypothesis that aberrations in kappa:lambda ratios early after allo-HSCT predict the occurrence of severe aGVHD.

Disclosures:
No relevant conflicts of interest to declare.