Background: The ROMA LA-OPSCC (NCT03759730) study prospectively evaluated the oral and gut microbiota in a single-centre cohort of LA-OPSCC patients (pts) receiving chemoradiotherapy (CRT). Methods: LA-OPSCC pts treated with definitive CRT (IMRT plus single-agent cisplatin) were eligible. Oral swabs over the tumor site and stool samples were collected at baseline and end of CRT (EOT). Taxonomic profiles were generated by 16S rRNA sequencing. ANOSIM/Kruskal-Wallis tests were used to identify differences between baseline and EOT samples. Results: A total of 96 samples were collected from 24 evaluable pts (100% compliance). Baseline characteristics: median age = 61 (range, 50-71); smoking status current/former/never = 5/11/8; HPV+/- = 23/1; stage I/II/III/IVA = 7/7/9/1; use of antibiotics = 12 pts. In oral swabs, decreased Shannon diversity (p< 0.01) and changes in abundance (adjusted p value: q< 0.05) of multiple taxa including Prevotella, Veillonella, andStreptococcuswere observed at EOT vs baseline. Stool diversity did not differ between baseline and EOT (p= 0.42), but abundance of Ruminoccocus and Roseburia decreased (q< 0.05). CRT-associated changes remained significant when controlled for stage, smoking, antibiotics, cisplatin dose and mucositis grade (p< 0.01). In HPV+ pts, stage I-II baseline oral swabs had higher relative abundance of Clostridium IV (p= 0.02) and Escherichia (p= 0.04) than stage III, which had higher Fusobacterium (p =0.03) and Gemella (p< 0.01). Relative abundance of Actinobacteria (p < 0.01), Proteobacteria (p < 0.01) andFirmicutes (p = 0.03) was higher in stool from stage III pts. Akkermansiamuciniphila was present in 57% of the stage I-II stool samples, and 11% in stage III (p= 0.04). Conclusions: CRT in LA-OPSCC is associated with increases in potentially pathogenic genera in the oropharynx. HPV+ stage III disease was associated with higher Fusobacterium in the oropharynx, which has been implicated in tumor metastases, and with decreased prevalence of the immunotherapy-response-associated species Akkermansia in stool. These preliminary observations suggest an opportunity for the evaluation of IO based therapies or manipulation of the gut microbiota in this patient population. Clinical trial information: NCT03759730