Background: Loss of argininosuccinate synthetase 1 (ASS1) sensitizes malignant pleural mesothelioma (MPM) and lung carcinoma (NSCLC) cells to apoptosis following arginine (L-Arg) deprivation. We showed that the L-Arg depletor pegylated arginine deiminase (ADI-PEG20, ADI) enhances the cytotoxic effect of pemetrexed (PEM) in ASS1-negative tumor cells involving suppression of pyrimidine synthesis and the salvage pathway. The latter was linked to inhibition of thymidine kinase 1 with reduced uptake of 18-FLT on PET imaging in vivo. In the phase I dose-escalation TRAP study (NCT02029690) we showed that ADI added to first-line PEM and cisplatin (CIS) chemotherapy (ADIPEMCIS) doubled the partial response (PR) rate in patients (pts) with ASS1-deficient MPM and NSCLC (Mol Cancer Ther 2015;14:B23). Here, we explored the role of 18-FLT-PET as a novel biomarker for assessing disease response to ADIPEMCIS. Methods: 18-FLT-PET was undertaken to assess tumor proliferation at baseline (scan 1), 24 hours post first dose of ADI and prior to PEMCIS on day 3 (scan 2), post first cycle ADIPEMCIS on day 15 (scan 3) and at end of ADIPEMCIS (scan 4) in 20 pts (10 pts each with MPM and NSCLC) since March 2015. Treatment response was measured using EORTC PET response criteria (PR, stable disease or SD, progressive disease or PD). A majority of pts have accrued to the PET imaging and updated results will be correlated with the RECIST responses. Results: Comparing with the baseline PET imaging in n = 11 pts: 2/9 (22%), 6/9 (67%), 1/9 (11%) had a PR/SD/PD on scan 1; 4/11 (36%), 6/11 (55%), 1/11 (9%) had a PR/SD/PD on scan 3; and 5/8 (63%), 3/8 (37%) had a PR/SD on scan 4. RECIST revealed PR = 5/9 (56%), SD = 3/9 (33%), and PD = 1/9 (11%) at end of treatment in the same group. Conclusions: 18-FLT-PET provides early evidence of response to L-Arg deprivation in pts with ASS1-deficient thoracic tumors. Furthermore, the rate of metabolic PRs is maintained and increases with more completed cycles of ADIPEMCIS. Our 18-FLT-PET studies confirm that L-Arg depletion impacts tumor proliferation as seen in the preclinical studies and warrants further investigation as a novel antimetabolite strategy with antifolate drugs for cancer therapy. Clinical trial information: NCT02029690