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High glycolitic activity of multiple myeloma (MM) cells is the rationale for the use of Positron Emission Tomography (PET) with 18F-fluorodeoxyglucose ([18F]FDG) to detect both medullary and extramedullary disease. However, FDG-PET has some limitations, since there is a good portion of MM patients who are false-negative. Besides enhanced glycolysis, glutamine (Gln) addiction has been recently described as a metabolic feature of MM by our group. To sustain high Gln demand, MM cells increase the expression of several Gln transporters (ASCT2, SNAT1, LAT1) and are endowed with fast Gln uptake. Yet, at variance with other Gln-addicted cancers, the possible exploitation of Gln as a PET tracer in MM has never been assessed and was investigated in this study. To this purpose, we have firstly synthesized enantiopure (2S,4R)-4-Fluoroglutamine (4-FGln) and validated it as a Gln analogue in human MM cell lines (RPMI8226 and JJN3) comparing its uptake with that of 3H-labelled Gln. The intracellular levels of 4-FGln were determined by HPLC-MS/MS employing a HILIC gradient separation and multiple reaction monitoring (MRM) detection. Both Gln and 4-FGln were actively accumulated by MM cells and exhibited a strong reciprocal competition, pointing to shared transporters. Inhibition analysis revealed that ASCT2 was the major entry route of both compounds, with minor contributions from the other transporters. However, compared with Gln, 4-FGln exhibited higher affinity for both ASCT2 and LAT1 transporters. On the basis of these results, we then tested [18F]4-FGln uptake for MM detection by Positron Emission Tomography (PET) in two different in vivo murine models. Firstly, to investigate sensitivity of human MM to [18F]4-FGln in vivo, JJN3 cells were subcutaneously injected in immunodeficient NSG mice In this xenograft model, [18F]4-FGln- and[18F]FDG-PET scans were performed after plasmacytomas became palpable and repeated after one week. All the tumours were positive for [18F]FDG and displayed [18F]4-FGln uptake with Standard Uptake Values (SUV) of 1.21±1.9 and 0.99±0.07 after 2 weeks, respectively. Thereafter, the effect of bortezomib (BOR) was investigated to evaluate the potential use of [18F]4-FGln to monitor anti-MM treatment. Ten NGS mice were injected with JJN3 cells and, after 14 days, treated twice weekly with BOR, 1mg/kg, or vehicle for two weeks. PET scans were performed before and after 5 and 12 days of BOR treatment. As expected, BOR reduced tumour size as compared to vehicle. At the first post-BOR PET scan, [18F]4-FGln (SUV mean: pre 0.85±0.31; post 0.45±0.10, P Thereafter, to mimic BOR-resistant MM in a syngeneic mouse model, C57BL/6 mice were injected intravenously with Vk12598 cells obtained from transgenic Vk*MYC mice repeatedly treated with sub-optimal doses of BOR. Upon injection into C57BL/6 mice, Vk12598 cells colonize the BM without lytic lesions and extensively colonize the spleen generating an aggressive MM that brings animals to death within five weeks. PET scans were performed with [18F]4-FGln and [18F]FDG before Vk*MYC MM cells injection and after three, four and five weeks. Blood samples for M-spike evaluation were obtained in parallel. Four weeks after MM cells injection a significant increase of both [18F]4-FGln and [18F]FDG uptake was detected in spleens (SUV mean: 1.14±0.23, P=0.018; 0.94±0.24, P= 0.005). In both MM models, the volume of distribution of [18F]4-F-Gln did not overlap that of [18F]FDG. In conclusion, our data indicate that [18F]-(2S,4R)-4-Fluoroglutamine is a new potential PET tracer in pre-clinical MM models especially of extramedullary disease, either in a BOR-sensitive or in a BOR-resistant context, supporting the exploitation of Gln addiction for diagnostic purposes in MM patients. Disclosures Giuliani: Janssen: Research Funding.