Background: Overexpression of the transmembrane oncoprotein HER2 occurs in 20-30% of invasive breast cancers and predicts for a poor clinical outcome. Trastuzumab, a monoclonal antibody against the extracellular domain of the HER2 receptor, importantly improves the prognosis of patients with HER2 positive breast cancer but primary and secondary resistance frequently occurs. The mechanisms of resistance are unclear, but are thought to involve a variety of signaling pathways including PI3K/Akt/mTOR and Ras/MAPK(ERK). Elucidating the mechanisms of trastuzumab resistance will enable to identify subgroups of patients who either will or will not benefit from the treatment with trastuzumab. In addition, an understanding of each mechanism can initiate the development of strategies to overcome resistance and ultimately improve the survival of women with HER2 positive breast cancer. In this study we performed an extensive immunohistochemical analysis of a variety of biomarkers possibly associated with trastuzumab resistance. Patients and methods: 155 patients diagnosed with primary HER2 positive invasive breast cancer diagnosed between 2003 and 2011 were included. Tissue microarrays (TMAs) were conducted from the original tumour tissue and immunohistochemistry was performed. A complete biomarker profile was constructed in 133 patients. We estimated the prevalence of overexpression of a variety of biomarkers and their association with patient outcome. Results: We found 70% ER positive-, and 51% PR positive tumours, overexpression of Ki67 (46%), p53 (47%), EGFR (11%), membranous IGF-IR (51%), cytoplasmic IGF-IR (55%), p-Akt (37%), p-mTOR (33%), p-P70S6K (42%), p-ERK (70%) and PTEN loss in 20% of the tumours. Our data demonstrate that various biomarkers are often simultaneously expressed. In this cohort, we found no significant difference in outcome between levels of any of the assessed markers. Conclusion: Our result support that simultaneous overexpression occurs of diverse biomarkers involved in the PI3K/Akt/mTOR, Ras/MAPK(ERK) and alternate receptor pathways, indicating coherence and cross-talk. Patterns of these biomarkers could be predictive of outcome in patients with HER2 positive breast cancer treated with trastuzumab. Unraveling patterns of predictive biomarkers is highly desirable for adequate patient selection for trastuzumab treatment and development of rational anti-tumour therapies. This study does not show prognostic significance for any of the biomarkers, probably due to the limited number of events. Therefore further studies on these markers in larger series are needed.