PurposeIn this study endogenous magnetic resonance imaging (MRI) biomarkers for accurate segmentation of High Intensity Focused Ultrasound (HIFU)-treated tumor tissue and residual or recurring non-treated tumor tissue were identified.MethodsMultiparametric MRI, consisting of quantitative T1, T2, Apparent Diffusion Coefficient (ADC) and Magnetization Transfer Ratio (MTR) mapping, was performed in tumor-bearing mice before (n = 14), 1 h after (n = 14) and 72 h (n = 7) after HIFU treatment. A non-treated control group was included (n = 7). Cluster analysis using the Iterative Self Organizing Data Analysis (ISODATA) technique was performed on subsets of MRI parameters (feature vectors). The clusters resulting from the ISODATA segmentation were divided into a viable and non-viable class based on the fraction of pixels assigned to the clusters at the different experimental time points. ISODATA-derived non-viable tumor fractions were quantitatively compared to histology-derived non-viable tumor volume fractions.ResultsThe highest agreement between the ISODATA-derived and histology-derived non-viable tumor fractions was observed for feature vector {T1, T2, ADC}. R1 (1/T1), R2 (1/T2), ADC and MTR each were significantly increased in the ISODATA-defined non-viable tumor tissue at 1 h after HIFU treatment compared to viable, non-treated tumor tissue. R1, ADC and MTR were also significantly increased at 72 h after HIFU.ConclusionsThis study demonstrates that non-viable, HIFU-treated tumor tissue can be distinguished from viable, non-treated tumor tissue using multiparametric MRI analysis. Clinical application of the presented methodology may allow for automated, accurate and objective evaluation of HIFU treatment.