Clinical Proteomics and Laser Capture Microdissection

Basic Sciences

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Abstract

In preliminary data derived with the Meso Scale system, (an ELISA based system) we detected differences in the cytokine expression of follicular lymphoma (FL) vs. follicular hyperplasia (FH). Whole follicle lysates from tonsillar FH (n=12), FL grade 1 (n=20), FL grade 2 (n=15) and FL grade 3 (n=12) were analyzed on a multiplex Meso Scale ELISA for detection of cytokine levels for IL-1&#61538;, IL-2, IL-4, IL-6, IL-8, IL-12p70, TNF alpha GM-CSF, IL-10, and IL5. In comparison to FL lysates, FH showed 25 times higher levels of IL-1 (p value < 0.0001), and 30 time higher levels of IL-8 (p value < 0.001). IL-12 was significantly higher in FH in comparison to FL G2 and G3 (p value < 0.03) but not significantly higher than FL G1. Conversely, IL-4 concentrations were over 20 times higher in FL grade 1 than in FH (p value = 0.01), and over 10 times higher in FL grade 2 than in FH (p value = 0.04). The difference in IL-4 concentrations between FL grade 3 and FH was not statistically significant (p value = 0.34). Interestingly, as the grade of FL increased, the overall IL-4 concentration decreased. IL-4 concentrations in FL G1 were over twice as high as in FL G3 (p value = 0.016). These results may reflect the inherent difference in the lymph node microenvironment between FL and FH. The statistically significant higher levels of IL-4 in FL grades 1 and 2, may suggest an important role for IL-4 in the tumor host microenvironment, or alternatively may merely reflect the presence of a higher number of tumor infiltrating T-cells in FL grade 1 and 2 relative to FH or FL grade 3. In order to determine the significance of elevated levels of IL-4 in FL grades 1 and 2, it will be useful to determine whether the IL-4 receptor is expressed on the tumor cells, whether the receptor and IL-4 dependent signaling pathways are activated, and whether activation confers a survival advantage to the tumor cells. The cytokine profiles characterized in the first component of the project represent a composite of the lymph node microenvironment. The next step will be to characterize the cytokine/chemokine receptor expression on individual subpopulations in FL. These profiles will be compared to profiles of B, T, and macrophage lineage cells in the follicles of FH and normal LN. Pure populations of the various hematopoietic constituents of FL, FH and normal LN will be generated using immuno-LCM on frozen sections of patient biopsies. Antibodies against CD20, CD3, and CD163 will be used to allow microdissection of pure populations from the heterogeneous populations in the native tissue biopsies. Subsequent analysis using RPMA will allow us to determine the quantitative immunophenotype of cytokine receptor expression in FL cells vs. B-cells in FH and normal LN. Likewise the cytokine receptor phenotypes of follicle T-cells and macrophages will be compared in FL vs. FH and normal LN. We believe these data will be important in understanding the mechanisms of immunotherapy, and in predicting the factors that lead to a successful response to monoclonal antibody based therapy.