IHC
tumor
TUMOR
For cohort allocation, HER2 status was determined by a GEFPICS trained pathologistRegarding T-DXd distribution during treatment, 10 paired baseline and on-treatment tumor biopsies were initially selected. Three pairs were not analyzed owing to the lack of tumor cells. Seven pairs of tumor biopsies, four from cohort 2 and three from cohort 3 (days 2–4 cycle 1, Multiplex immunofluorescence was performed with the Ultivue kit containing the Immuno8 FixVUE panel composed of eight pre-diluted antibodies (twice four barcoded markers) + DAPI ready to use. The antibodies were directed against CD3 (clone BC33), CD4 (clone SP35), CD8 (clone C8/144B), CD68 (clone KP-1), FoxP3 (clone 236A/E7), PD-1 (clone CAL20), PD-L1 (clone 73-10) and PanCK/SOX10 (clone AE1/AE3/BC34). Thirty-one paired FFPE tumor biopsies obtained at baseline and on days 22–43 after cycle 1 were stained (18 cohort 1, 10 cohort 2, three cohort 3; Extended Data Fig. 