Background: Pretreatment (preRx) biopsies of HER2-amplified breast cancers (BCs) that did not achieve pCR with preop chemotherapy plus H vs L vs HL had PI3K pathway activation with high p-FOXO levels on RPMA (Holmes BMC Res Notes 2013). The RD obtained at surgery in these pts was enriched for co-mutations in PIK3CA and TP53 (Holmes ASCO 2014, 625). Here we describe activated phosphoproteins in the HER1/2/3 pathway in the RD from these pts who did not achieve pCR with preop H vs L vs HL and in RD with PIK3CA/TP53 co-mutations. Methods: 15 pts’ FFPE RD BCs were acceptable for RPMA at a CLIA-certified laboratory (Theranostics Health). Immunostaining with 14 antibodies was directed against HER1/2/3 pathway proteins. Spearman correlation (ρ, p value) and Mann-Whitney U tests (p value) were performed. Results: 7 of 15 RD BCs had PIK3CA/TP53 co-mutations. In the RD with co-mutant PIK3CA/TP53, the strongest Pearson correlations (r) were between HER3 and p-HER1 and each with p-mTOR, p-S6K, p-JAK2 and p-STAT3 (all r > 0.9) In preop L pts (n = 8), the strongest correlations in the RD were p-HER2 and HER3, p-HER2 and p-mTOR, p-HER1 and p-STAT3 , p-HER3 and p-STAT3 (all r > 0.8), and p-4EBP1 and p-JAK2 (r = 0.97). In preop H and HL pts (n = 7), the RD showed strong interactions between HER3 and p-HER1, p-HER2 and p-HER3, HER3 and p-S6K, p-HER1 and p-S6K and p-HER3 and p-JAK2 (all r > 0.9). Comparing L- vs H/HL-treated RD, L-treated RD had higher levels of p-Akt (p = 0.08), p-S6 (p = 0.08) and p-MEK1/2 (p = 0.02). PreRx biopsies from pts who had RD all expressed high PI3K or low PTEN ser380 levels and showed strong interactions between p-HER1 and p-IGF1R, p-HER1, PI3K, and MAPK activation, LCB3 (autophagy) and PI3K, and Musashi (stem cell regulator) and β-catenin (all ρ > 0.8) (all p < 0.00006). Conclusions: PreRx biopsies from HER2+ pts who had RD following preop chemotherapy + H, L or HL showed activation of HER1, PI3K and MAPK pathways. Pts’ refractory RD demonstrated HER3, PI3K and JAK2 pathway activation under the selective pressure of preop H, L or HL. PIK3CA/TP53 co-mutated RD demonstrated signaling via p-HER1/HER3 to p-mTOR/p-S6K and p-JAK2/p-STAT3.