Aim 4. Immune signatures validation in patient biopsies

Once we establish the immune signatures for each clinical group (Aim 3), we need to validate our findings. This validation will be performed on formalin-fixed and paraffin-embedded (FFPE) human tumor tissues from biopsies from FL patients belonging to the previously defined groups, using two techniques: conventional immunohistochemistry (IHC) and multiplex-immunofluorescence.

Importantly, both techniques are based in the detection of proteins in the tumor, while in the generation of the signatures we will use an RNA-based method. As explained in Aim 2, RNA reflects the genes that are actually been expressed in the cells. However, the phenotype of the cell is determined by proteins, which are synthesized from the RNA. Furthermore, tissue imaging provides spatial information of how different populations are organized within the tumor microenvironment. Therefore, by validating our findings using another approach, we ensure that our results are robust and the signatures will have higher probability to be translated into the clinic.

Conventional IHC has been a widely used technique for many decades, in both Pathology units for diagnosis and in research platforms. In recent years, multiplex-immunofluorescence has gained growing attention, since it allows for the detection of multiple proteins. The group of Dr. Carlos de Andrea (Clínica Universidad de Navarra) has demonstrated expertise in this field and developed and validated this technique in glioblastoma, lung cancer and melanoma, in the context of immunotherapy studies.