Background: Pre-clinical data support a hypothesis that DNA methyltransferase inhibition will circumvent cisplatin resistance in various cancers including urothelial carcinoma (UC). SPIRE comprised a previously reported phase Ib dose escalation phase for incurable metastatic solid cancers which established a recommended phase II dose (RP2D) for guadecitabine combined with gemcitabine and cisplatin (GC) chemotherapy (Crabb et al, ESMO Congress 2018, abstract 425P). We now report the SPIRE phase IIa randomised dose expansion phase which tested neoadjuvant treatment of bladder UC. Methods: Patients had T2-4a N0 M0 bladder UC intended for radical treatment. All patients received an investigator choice of 3 or 4 planned, 21-day, GC cycles (cisplatin 70 mg/m², IV, day 8; gemcitabine 1000 mg/m², IV, days 8 and 15). 20 patients were randomised (1:1, open label) to whether they also received guadecitabine 20 mg/m², SC, on days 1 to 5, and G-CSF prophylaxis 300 µg, SC, on days 15 to 21. The primary objective for the expansion phase was to confirm a safe and biologically effective dose and schedule for this combination for future investigation. Circulating cell free DNA LINE-1 promotor methylation was measured as a guadecitabine pharmacodynamic endpoint. Trial registration: ISRCTN 16332228. Funding: Cancer Research UK, Astex Pharmaceuticals. Sponsor: University Hospital Southampton NHS Foundation Trust. Results: Median age was 68 (interquartile range (IQR) 59-72). 19 (95%) patients were male and 17 (85%) had T2 stage. The commonest grade ≥3 adverse events were neutropenia and thrombocytopenia with one or both affecting 6 (60%) patients in each treatment arm (no grade 5 events). One episode of neutropenic fever occurred (guadecitabine arm). Addition of guadecitabine to GC, versus GC alone, resulted in similar cisplatin dose intensity (median total doses 408 mg (IQR 384-435 mg) and 435 mg (IQR 384-435 mg) respectively) but modestly reduced gemcitabine dose intensity (median total doses 10,450 mg (IQR 9,500-11,400) and 12,768 mg (IQR 9,500-12,768) respectively). All patients completed post-chemotherapy radical treatment (8 cystectomy, 2 radiotherapy, in each arm) with similar timing post chemotherapy and peri-operative morbidity scores. LINE-1 promotor methylation depletion occurred at cycle day 8 in guadecitabine treated patients. Conclusions: Guadecitabine in combination with GC and G-CSF is safe and tolerable in this combination compared to GC alone as neoadjuvant treatment for UC. Radical surgery or radiotherapy delivery, and cisplatin dose intensity, were not compromised. Pharmacodynamic endpoints are optimal with this treatment schedule. Addition of guadecitabine to GC warrants further investigation. Clinical trial information: 16332228.