Quantifying Tumor Immune Infiltrates

Tumors were embedded in formalin and fixed in paraffin (FFPE) and sectioned into 4 μm thick slices and mounted onto slides. Slides were then stained for CD3 (clone CD3-12, AbD Serotec, Raleigh, North Carolina), CD8 (4SM15 eBioscience, Waltham, MA), pS6 (Polyclonal, Cell Signaling Technology), and DAB and counterstained with hematoxylin before being coverslipped. Slides were then scanned with an Aperio Slide Scanner and quantified digitally by measuring the average of 5 representative 1 mm² regions within the tumor to get a score in counts/mm².^{54 (link)}

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White M.G., Szczepaniak Sloane R., Witt R.G., Reuben A., Gaudreau P.O., Andrews M.C., Feng N., Johnson S., Class C.A., Bristow C., Wani K., Hudgens C., Nezi L., Manzo T., De Macedo M.P., Hu J., Davis R., Jiang H., Prieto P., Burton E., Hwu P., Tawbi H., Gershenwald J., Lazar A.J., Tetzlaff M.T., Overwijk W., Woodman S.E., Cooper Z.A., Marszalek J.R., Davies M.A., Heffernan T.P, & Wargo J.A. (2021). Short-term treatment with multi-drug regimens combining BRAF/MEK-targeted therapy and immunotherapy results in durable responses in Braf-mutated melanoma. Oncoimmunology, 10(1), 1992880.

Publication 2021

CD3 (clone CD3-12,

Clone Formalin Hematoxylin Paraffin Tumor

Corresponding Organization : The University of Texas MD Anderson Cancer Center

Other organizations : Olivia Newton-John Cancer Wellness & Research Centre, La Trobe University, Nektar Therapeutics (United States)

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Variable analysis

independent variables

Tumor embedding (formalin and paraffin)
Tissue sectioning (4 μm thick slices)

dependent variables

CD3 expression
CD8 expression
PS6 expression

control variables

Slide mounting
Staining (DAB and hematoxylin)
Coverslipping
Slide scanning (Aperio Slide Scanner)
Quantification (average of 5 representative 1 mm^2 regions within the tumor)

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