Background: Dual HER2 blockade with LT without chemotherapy resulted in a substantial pathologic complete response (pCR) rate in HER2+ BC patients (pts) (TBCRC006). High levels of tumor infiltrating lymphocytes (TILs) at diagnosis have shown to be associated with greater benefit to T and chemotherapy in early-stage HER2+ BC pts. However the predictive value of TILs, TILs’ subsets, and other immune cells in pts receiving anti-HER2 treatment alone without chemotherapy is still unknown. Methods: Pre-treatment biopsies from pts enrolled in the TBCRC006 trial were used to investigate tumor immune infiltrate at diagnosis. Hematoxylin and eosin (H&E)-stained slides were evaluated as currently recommended (Ann Oncol 26: 259, 2015) for the % of stromal TILs; a threshold of 60% was used to define lymphocyte-predominant BC (LPBC). Single formalin-fixed paraffin-embedded slides from 10 cases were co-stained for CD4, CD8, CD20, CD68, FoxP3, cytokeratin, and DAPI by multiplexed immunofluorescence (m-IF) using PerkinElmer Opal system. Multispectral imaging and digital analysis to visualize and quantify specific immune infiltrates are being performed using PerkinElmerVectra system. Results: TILs evaluation was available for 59 of the 64 pts who were evaluable for pathologic response. Twelve of 59 pts (20%) exhibited a LBPC. The pCR rate was numerically higher in LBPC compared to non-LPBC (50% vs. 19%, P = 0.057). Multispectral imaging successfully captured and quantified multiple immune cell types in all the so far m-IF-stained samples. The density of stromal TILs (calculated as the total number of cells positive for the above immune markers/mm2) significantly correlated with % of stromal TILs evaluated on the H&E-stained slides (r = 0.76, P = 0.01). Conclusions: LBPC was associated with numerically higher pCR rate than non-LPBC in patients with HER2+ BC treated with anti-HER2 agents without chemotherapy. However, P-value did not reach statistical significance. Characterizing TILs with m-IF is feasible and may help correlate various TIL subpopulations with response to treatment. Complete results for the entire cohort will be presented at the meeting.