The HER2 splice variant lacking exon-16 (d16HER2) has been detected in human breast carcinomas.It has been shown in proper transgenic mouse models that d16HER2 variant leads to an increased transforming potency compared to the wild-type (wt) HER2 receptor¹. Magnetic Resonance imaging (MRI) and Spectroscopy (MRS) have demonstrated their usefulness in the tumour diagnosis, prognosis and in the evaluation of in vivo therapy. In this work, a murine mammary carcinoma cell line transgenically expressing the human d16HER2 variant (MI6 cells)-was implanted in the mammary fat pad of parental FVB mice1 which were treated with lapatinib or phenethyl isothiocyanate (PEITC), two different anti-tumor coumpounds respectively targeting HER2 receptor² and breast cancer initiating cells³ , and analyzed by in vivo quantitative MRI and MRS analyses.This work was aimed to explore the possibility to non-invasively detect metabolic, morphologic and functional alterations in a d16HER2 mammary carcinoma in vivo model during different treatments.FVB mice were injected with 1x10⁶ of MI6 cells in thoracic mammary fat pad¹. Fifteen days post injection animals were randomly allocated to three groups and treated per os for 5 days a week for 11 weeks with: 1) lapatinib (100 mg/kg, n=5); 2) PEITC, (1.8 mg/kg, n=5); 3) DMSO as control n=6). In vivo MRI/MRS analyses were performed by using an Agilent Inova MRI/MRS system (4.7 T) between 68 and 88 days post tumor injection. MRI evaluation was performed by T1W (TR/TE=400/7 ms), T2W (TR/TE=3000/70ms) and DW (TR/TE=2500/50 ms, b-value ranging between 0 and 1100 s/mm²) multislice spin echo images. D, f and D* parameters were derived from DWI by applying IVIM analysis. Quantitative 1H MRS analyses were performed by following a quantitative protocol described in ⁴ (PRESS TR/TE = 4000/23 ms) which include water T2 measurements and which assume 80% of tumour water content. LCModel was used for the spectral fitting. Ex vivo MRS analyses were performed on tissue extracts at 9.4 and 16.4 T by using high resolution Bruker Avance spectrometers as described by⁵.The administration of Lapatinib and PEITC led to a significant tumor growth delay in treated mice compared to controls. Despite a delay in tumor growth revealed in Lapatinib and PEITC groups (Fig. 1), different internal tumor composition has been detected by MRI (Fig. 2). In fact, PEITC-treated tumours (Fig. 2b) exhibit more heterogeneity with respect to the controls (Fig. 2a) and Lapatinib-treated tumours (Fig. 2c), i.e. the presence of small and widespread hyper- and hypo-intense regions in the T2-weighted and diffusion-weighted images. The heterogeneity in the T2-weighted is likely due to the presence of haemorrhage (hypo-intense areas) or necrosis (hyper-intense regions)⁶. Quantitative DWI and T2 analyses showed differences among the groups (Fig 3). None of them reach a statistical significance due to the limited number of animals per group. It is interesting to note the high perfusion value (D*) in the PEITC group (obtained with IVIM analysis). Quantitative metabolic analyses are summarised in Fig.4. In all treated tumours we found an increase in lipid signals and a decrease in m-Ins content, being the latter a fingerprint of d16HER2 spectrum when compared with the human WT HER2 expressing mammary tumors.These preliminary results suggest MRI/MRS as suitable approaches to investigate the effect of Lapatinib- and PEITC-based therapies in addition to the measure of tumor volume , and encourage further investigations with increased number of animals and correlations with biological, histological and immunohistochemical analyses, which are ongoing.