IHC staining and interpretation of CCR4

CCR4 IHC tissue staining was performed using a mouse anti-human CCR4 monoclonal antibody (clone L291H4; Biolegend #359402) at 1.25 ug/ml. Staining was performed on 4 μm formalin-fixed, paraffin-embedded tissue sections. Tissue sections were baked for one hour at 62⁰C and dewaxed on the Leica Bond. Antigen retrieval was performed in Leica ER2 solution for 30 minutes followed by 30 minutes of primary antibody incubation at room temperature. Antibody deposition was reported using DAB (Bond Polymer Refine Detection). CCR4 stained sections were reviewed by three of the coauthors. For each case, the percentage of the atypical lymphoid infiltrate positive for CCR4 was evaluated by a reviewer who was blinded to the clinical diagnosis. CCR4 staining intensity was graded 1+, 2+ or 3+. The size and location of CCR4-positive cells and staining pattern ((I) granular or diffuse and (II) cytoplasmic, perinuclear or membranous, were noted. Positive cases were subsequently stratified into two categories based on the percent of CCR4+ cells of the whole infiltrate using a natural cut point of 25%: low expression if 0–25% and high expression if >25%.¹² H-score was calculated for each case by multiplying the percent of CCR4+ cells by the intensity grade.