CD86 is highly expressed in primary diagnosis and R/R cHL. (A) Summary of treatment schedules and cohort distribution of the NIVHAL trial.⁵² (B) Heat map illustrating expression of reference control gene CD30 or candidate antigens CD86, CD80, and PD-L1 in the NIVHAL cohort (n = 95 primary diagnosis samples). Each line represents 1 patient. (C) Change of target antigen expression between primary (gray) and secondary (light green, red) biopsies after NIVHAL trial first-line therapy. Treatment response was evaluated by a board-certified pathologist and classified into HRSC clearance (light green, n = 4 primary patient samples) or HRSC maintenance (red, n = 6 primary patient samples). Statistical significance was calculated using 2-way ANOVA with Sidak multiple comparison correction. (D-E) H&E (left) or immunohistochemical staining of CD86 (middle) in comparison to CD30 (right) in primary cHL tissue. (D,F,G) Primary diagnosis cHL samples. n = 7 different patients. (E,H-I) R/R cHL samples. n = 10 different patients. (F,H) Representative images of CD86⁺ HRSCs. Arrowheads indicate HRSCs. (G,I) Count of CD86⁺ or CD30⁺ HRSCs in primary diagnosis cHL samples (G) or R/R cHL (I) quantified by machine learning classifier. Statistical significance was calculated using unpaired t test. (J-K) Expression of CD86 on HRSCs measured by multiplex immunofluorescence microscopy on chip-loaded primary diagnosis cHL samples (J) or R/R cHL (K). Chips were sequentially stained with antibodies against CD86 (yellow), CD30 (blue), and CD20 (red) and with a DNA Hoechst stain (blue). Between each staining step, images were acquired with a fluorescence microscope followed by a 30-second photobleaching procedure. For all panels: ∗P < .05; ∗∗P < .01; ∗∗∗P < .001; ∗∗∗∗P < .0001. ns, P > .05.