Advice for design matrix

[bl24]

Hi, I'm working on my first GeoMx dataset and I'm looking to make a comparison that's more complex than I'm used to dealing with.

The design is that I have four ROIs: 1-4, 1 being my normal and 2 being my cancer data. I have two cancer stages within the ROI2 that I want to compare, T2 and T3. But before doing that comparison, I wanted to somehow adjust the variation in my ROI2 data using the ROI1 normal. My confusion is whether or not this can be done using one design matrix or do I need to do the ROI2/ROI1 comparison, extract the residuals and then perform the comparison on that?

Here's a screenshot of the Annotation file for the data: