Circulating tumor DNA (ctDNA) for response assessment in patients with anal cancer treated with definitive chemoradiation.
Authors: Alvarez Janet , Cercek Andrea , Mohan Natasha , Cuaron John J , Zinovoy Melissa , Reyngold Marsha , Yaeger Rona , Hajj Carla , Fanta Corrine , Wong Charles , Segal Neil Howard , Paty Philip , Crane Christopher H. , Garcia-Aguilar Julio , Weiser Martin R. , Smith Jesse Joshua , Tuli Richard , Romesser Paul Bernard 
Published: January 2023

1 Background: We hypothesized that circulating tumor DNA (ctDNA) clearance could provide an early signal for clinical complete response (cCR) and/ or long-term recurrence compared to standard clinical exam modalities in patients with anal squamous cell carcinoma (ASCC) undergoing definitive chemoradiation (CRT). Methods: Since early 2021 patients with ASCC undergoing CRT at 2 institutions were offered ctDNA monitoring with a commercially available tumor-bespoke multiplex PCR assay. All patients provided written informed consent for ctDNA testing. Patients were clinically restaged, 3-4 months post-CRT, by clinical exam, endoscopy, and/ or MRI and annually with CT chest, abdomen, and pelvis. cCR was defined as no tumor by digital exam, endoscopy and/or MRI. Molecular ctDNA response is described according to cCR, tumor recurrence, and survival. Results: From January 2021 to September 2022, 31 patients with ASCC treated with definitive CRT underwent ctDNA response assessment. The majority (68%) of patients had stage III disease. Patients were treated to a median radiation dose of 54Gy in 27 fractions with combinatorial mitomycin and fluoropyrimidine-based chemotherapy in 94%, and fluoropyrimidine-based chemotherapy alone in 6%. The median follow up was 22 weeks. ctDNA testing was performed in 25 of these patients at baseline, 26 patients during CRT, and 20 patients 30-days post-CRT. At baseline 88% of patients had detectable ctDNA. Patients with stage III, as compared to stage I-II, disease had numerically higher baseline ctDNA levels (26 vs 4 mean tumor molecules per milliliter (MTM/mL), p=0.08). ctDNA levels decreased with treatment (19 vs 0.9 MTM/mL, p=0.05) among the 18 patients with detectable ctDNA and ctDNA tested during CRT, with 50% of patients entering molecular remission. Similarly, ctDNA levels decreased (21 vs 0.2 MTM/mL, p=0.05) among the 16 patients with detectable ctDNA and ctDNA tested post-CRT, with 94% entering molecular remission. All patients in molecular remission were confirmed to have a cCR. Time to molecular ctDNA remission was significantly shorter than time to cCR (median 30 vs 135 days, p <0.01). There were no molecular recurrences among the 16, 14, and 7 patients with ctDNA testing at 2-4 months, 4-8 months, and 8-12 months post-CRT. All patients are alive and without clinical/ radiographic evidence of disease. Conclusions: Surveillance ctDNA monitoring may provide an earlier response assessment for patients with ASCC undergoing CRT compared to standard clinical measures. Longer term follow-up is required to determine if ctDNA response correlates with long term recurrence free survival. Larger trials are needed to assess the clinical utility of integrating molecular ctDNA response in therapeutic response surveillance.