MiRNAs have been suggested as promising biomarkers, including potential markers of Tamoxifen resistance. Herein we report the first large-scale global miRNA expression study on primary tumors from high-risk, ER+, post-menopausal breast cancer patients who received adjuvant Tamoxifen mono-therapy. Our study showed that although a highly significant set of 10 miRNAs distinguishing patient samples according to outcome were identified in the discovery set, the miRNA profile could not be confirmed in the subsequent two independent test sets. Our data indicate that there is likely no single, strongly predictive profile of outcome, and even small imbalances with regard to prognostic factors between the recurrent/non-recurrent groups, such as number of tumor-infiltrated lymph nodes at time of diagnosis, may mask identification of potentially predictive miRNAs. Further, it may be speculated that endocrine resistance occurs as a result of several different mechanism, each employing a set of unique miRNA, complicating the identification of a common set of miRNAs that is altered in primary tumors of breast cancer patients who do not have the benefit of adjuvant Tamoxifen treatment. This finding is somewhat surprising, since several miRNAs have been suggested to be associated with Tamoxifen resistance. However, the majorities of these studies were performed in cell line models or used very limited numbers of patient samples. Moreover, several studies identified miRNAs involved in ER regulation, and thus only indirectly proposed to be involved in Tamoxifen resistance. One of these, the miR-221/-222 cluster, has been found to be in a feedback loop with ERa that results in a decrease in ERa protein, which can lead to resistance towards Tamoxifen. The reason for not identifying this miRNA cluster in our study may be related to the fact that most of the patient tumor samples we analyzed exhibited a very high percentage of ERa positive cells, with a median of 83%, across the three sets. Another miRNA, miR-210, was identified in a dataset of untreated patients consisting of 25 ER-, 40 ER+ and 8 ERunknown tumor samples, and therefore likely represents a general marker for outcome, whereas our study focused on resistance towards Tamoxifen from the onset. Another recent large study could also not confirm the association of miR-210 with outcome. The discovery set was clinically very homogenous, and the Nand R-groups were nearly identical according to the strong prognostic factors that influence the likelihood of developing recurrence. The two test sets, on the other hand, had more variable clinical characteristics per group. Where the R-group had an average of 8.6, whereas the N-group had 3.0. For test set #2, there was a minor difference in the average tumor size and in tumorinfiltrated lymph nodes, but there was also variation in the duration of treatment with Tamoxifen. The two test sets were randomly selected, as opposed to the discovery set, which was matched, and potential biomarkers, such as miRNAs, would need the capacity to overcome patient-to-patient variation to achieve clinical value. Thus, the miRNA profile does not seem to provide information with regard to the probability of GSK2118436 1195765-45-7 recurrence following adjuvant Tamoxifen-treatment in post-menopausal ER breast cancer patients.