Background: 5-FU and mitomycin-C (MMC) with radiotherapy (RT) remain an established treatment for pts with LA (T2-4, N0-1, M0 or T1-4, N2-3, M0) AC. Genetic mutations in 2 major metabolizing enzymes for 5-FU; DPYD and TYMS have been associated with clinical response and toxicity. However their place in treatment of AC remains undetermined. Methods: We retrospectively reviewed 21 pts with LA AC treated between 2012 - 2018. All pts were treated with 5-FU 1,000mg/m²/day continuous IV infusion 1–4 and 29–32, MMC 10mg/m² IV bolus Days 1 and 29 plus RT. Acute toxicity was recorded and discussed during weekly multidisciplinary meetings. The worst grade was scored from start of treatment until 30 days after the last fraction of radiotherapy according to the NCI-CTCAE, v4.03. Tumor response was evaluated by DRE and palpation of inguinal nodes during treatment, at the end of treatment, and radiological imaging 4–6 weeks after completion of treatment. Pts who developed ≥3 toxicities were tested for DPYD and TYMS genes (2 major polymorphisms has been associated with altered TYMS: polymorphic 28bp tandem repeat polymorphism in 5’-untranslated region (5’UTR) into TYMS sequence enhancer region (TSER) and TYMS 1494del, is a 6-base pair (bp) deletion polymorphism in 3’-UTR. Results: 6/21 ptsdeveloped severe toxicities (Caucasians; 5 females, 1 male; age range: 42 -68 yrs) consisting of grade ≥3 pancytopenia, neutropenia, thrombocytopenia, mucositis, nausea, rash and nephritis. The most common genetic mutations found in these pts included TYMS 2RG/3RC (2), 3RG/3RC (1), 2R/2R (2), TYMS 3 ' UTR del/Ins (2) and DPYD c.2864A>T heterozygous (1). Treatment was changed in 2 pts to MMC 10 mg/m² Day 1 and 29 with cisplatin 25 mg/m²/week plus RT. These 2 pts reached pCR at 70 days and other 4 pts at 140 days. Conclusions: Molecular testing for DPYD and TYMS genes can allow us to identify pts who are most likely to respond or face severe toxicity to 5-FU. Combining radiation with MMC and cisplatin in pts with LA AC is feasible and EORTC is currently comparing this combination with MMC/5-FU in a large phase III trial.