we performed whole exome sequencing of pretreatment biopsies and examined whether genome wide metrics of overall mutational load clonal heterogeneity or alterations at variant gene and pathway levels are associated with treatment response and survival two hundred and three biopsies from the neoaltto trial were analyzed mutations were called with mutect and strelka using pooled normal dna associations between dna alterations and outcome were evaluated by logistic and cox proportional hazards regression there were no recurrent single gene mutations significantly associated with pathologic complete response pcr except pik3ca odds ratio or 0 42 p 0 0185 mutations in 33 of 714 pathways were significantly associated with response but different genes were affected in different individuals pik3ca was present in 23 of these pathways defining a trastuzumab resistance network of 459 genes cases with mutations in this network had low pcr rates to trastuzumab 2 50 4 compared with cases with no mutations 9 16 56 or 0 035 p 0 001 mutations in the regulation of rhoa activity pathway were associated with higher pcr rate to lapatinib or 14 8 adjusted p 0 001 lapatinib trastuzumab or 3 0 adjusted p 0 09 and all arms combined or 3 77 adjusted p 0 02 patients n 124 with mutations in the trastuzumab resistance network but intact rhoa pathway had 2 1 41 pcr rate with trastuzumab alone or 0 026 p 0 001 but adding lapatinib increased pcr rate to 45 17 38 or 1 68 p 0 3 patients n 46 who had no mutations in either gene set had 6 pcr rate 1 15 with lapatinib but had the highest pcr rate 52 8 15 with trastuzumab alone mutations in the rhoa pathway are associated with pcr to lapatinib and mutations in a pik3ca related network are associated with resistance to trastuzumab the combined mutation status of these two pathways could define patients with very low response rate to trastuzumab alone that can be augmented by adding lapatinib or substituting trastuzumab with lapatinib.