Abstract
Patients with anal squamous cell carcinoma (ASCC) who fail chemoradiation (CRT) have poor outcomes, underscoring the need for biomarkers to guide risk stratification. In a real-world two-center cohort of 84 adults with non-metastatic ASCC treated with curative-intent CRT, we prospectively evaluate a tumor-informed circulating tumor DNA (ctDNA) assay (SignateraTM, Natera). Here we show that across 647 plasma specimens, ctDNA is positive at pre-treatment in 79% (61/77), including 89% (24/27) with stage III disease. End-of-treatment ctDNA positivity identifies patients with inferior one-year outcomes: 63% overall survival, 44% progression-free survival, and 39% locoregional failure. Conversely, patients who were ctDNA-negative at baseline or who cleared ctDNA during-treatment have 100% locoregional failure-free survival. During surveillance, ctDNA re-emergence precedes clinical or radiographic relapse in every case. These findings support the consideration of ctDNA as a dynamic, treatment-responsive biomarker warranting prospective validation for risk-adapted surveillance and adjuvant therapy in ASCC.
Data availability
All data necessary to interpret the findings are presented in the article (Fig. 1 shows patient-level mutational profiles; Fig. 2 shows patient-level courses, outcomes, and ctDNA results for all patients; Fig. 5 shows ctDNA profiles, clinical and radiographic response assessments, and details on treatment failures for the seven patients with molecular recurrence; Supplementary Fig. 2 shows ctDNA profiles, clinical and radiographic response assessments, and details on treatment failures for all patients with treatment failure or death). Source data underlying Figs. 3, 4, and 5 alongside Supplementary Figs. 1 and 2 are provided with this paper in the Source Data file (see README tab for sheet map and variable definitions). De-identified clinical/ctDNA datasets (sample IDs, time-stamped results, MTM/mL) and underlying raw sequencing files are available under restricted access because public deposition is not permitted by IRB approvals, participant consent, HIPAA privacy regulations, and institutional/contractual obligations. Access can be obtained by emailing the corresponding author (P.B.R.; romessep@mskcc.org) with a brief research proposal, institutional affiliation, and IRB/ethics documentation (or a non-human subjects determination). Requests will be acknowledged and reviewed within 2–3 weeks; upon approval, a data transfer/use agreement with Memorial Sloan Kettering Cancer Center, coordinated as needed with the University of South Florida and Natera, will be executed and data shared via secure transfer for non-commercial verification/replication only, with no redistribution or secondary use, for the duration specified in the agreement. No external datasets were used in this study. Source data are provided with this paper.
Code availability
Analyses were performed in R (v4.4.0) using openly available packages (tidyverse v2.0.0, gtsummary v2.0.0, ggsurvfit v1.1.0, ggalluvial v0.12.5, ggpubr v0.6.0). No novel algorithms were developed. Custom scripts used for data wrangling, survival analyses, and figure generation are available under restricted access; requests to the corresponding author (romessep@mskcc.org) will be reviewed within 2–3 weeks and, if approved, shared under a data transfer/use agreement for non-commercial verification only (no redistribution or secondary use).
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Acknowledgements
This work was supported in part by National Institutes of Health/National Cancer Institute (NIH/NCI) Memorial Sloan Kettering Cancer Center (MSK) Support Grant [P30 CA008748]. Dr. Smith is salso upported by an NIH/NCI grant [R37 CA248289]. Dr. Romesser is also supported by an NIH/NCI grant [K08 CA255574] and a NIH/NCI grant (R37 CA304010). Dr. Bercz is also supported by an NCI Surgical Oncology T32 Research Training Grant [5T32 CA 9501-34]. This work was presented as an oral podium presentation at the 2025 ASCO Annual Meeting in Chicago, IL, USA. The authors would like to acknowledge the entire Colorectal Disease Management Team at Memorial Sloan Kettering Cancer who provided exceptional care to these patients. The authors acknowledge the assistance of ChatGPT in generating initial editorial suggestions and thank Jennifer Huber, PhD and Charuta Palsuledesai, PhD for their editorial support. The authors would also like to thank Robert Lentz, MD and Kathryn Winter, PhD for their thoughtful review of the manuscript.
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Authors P.B.R., A.B., J.A., C.E.K., M.G., J.J.S., and R.T. had full access to all the data in the study and take responsibility for the integrity of the data and the accuracy of the data analysis. Concept and Design: P.B.R. Acquisition, analysis, or interpretation of data: P.B.R., A.B., J.A., C.E.K., Y.J.H., M.G., J.J.S., and R.T. Drafting of Manuscript: P.B.R., A.B., J.A., R.T. Critical Revision of the manuscript for intellectual content: P.B.R., A.B., J.A., C.E.K., A.A., E.K.L., Y.J.H., N.M., M.R., R.Y., D.A.R.O., J.J.C., M.Z., C.O., R.R., J.L., W.Z., V.N.A., S.S., M.M., S.R.H., N.S., A.J., M.C.L., A.W., V.W., L.C., E.P., D.R., N.H.S., P.B.P., M.R.W., A.C., J.M., J.G.A., C.H.C., M.H., J.J.S., R.T. Statistical Analysis: P.B.R., A.B., J.A., C.E.K., Y.J.H., M.G. Administrative, technical, or material support: P.B.R., M.R.W., P.B.P., M.G., J.G.A., J.J.S., R.T. Supervision: P.B.R.
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Dr. Romesser provides compensated professional services and activities for EMD Serono, Faeth Therapeutics, HPV Alliance, and Natera Inc. He also offers uncompensated professional services and activities for 10x Genomics, XRad Therapeutics, and the HPV Alliance and Anal Cancer Foundation non-profit organizations. Dr. Smith received travel support for fellow education from Intuitive Surgical (August 2015). He also served as a clinical advisor for Guardant Health (March 2019). He provides compensated professional services and activities for Johnson and Johnson, GlaxoSmithKline, Foundation Medicine, UroGen, Regeneron, and Vaniam Group. Dr. Garcia-Aguilar owns equity and received honoraria from Intuitive Surgical. Dr. Weiser owns intellectual property rights and provides compensated professional services for UpToDate. Dr. Cercek owns equity in Haystack Oncology, Inc. She also provides compensated professional services for AbbVie, Agenus, Amen, Daiichi Sankyo, GlaxoSmithKline, Illumina, Janssen Oncology, Inc., Merck & Co. Inc., Regeneron Pharmaceuticals, Inc., Roche Diagnostics Asia Pacific Ltd., and Seagen. Dr. Crane owns equity in Oncternal Therapeutics and provides compensated professional services for Trisalus Life Sciences. Dr. Segal provides compensated professional services for Puretech Health, Regeneron Pharmaceuticals Inc., and Agenus Inc. Dr. Reyngold provides compensated professional services and activities for Elekta and offer uncompensated professional services for the National Comprehensive Cancer Network. Dr. Yaeger provides compensated professional services and activities for Lilly Oncology, Mirati Therapeutics, Revolution Medicines, and Merck. Dr. Wu provides compensated professional services and activities for CivaTech Oncology, Inc., MORE Health, Inc., Nanovi A/S, and Simphotek, Inc. Dr. Connell provides compensated professional services and activities for Intera Oncology, Inc. Dr. Rao owns equity in Abbott Laboratories, CVS Health Corp., GlaxoSmithKline, Merck & Co., Novartis Pharmaceuticals Corporation, Procter & Gamble, Sandoz, Inc., Sanofi-Aventis U.S. LLC, and UnitedHealth Group. Drs. Aushev, Sharma, Malhotra, Rivero-Hinojosa, Jurdi, and Liu are employees of Natera, Inc. and receive equity in the company. All other authors do not have any conflicts of interest to disclose. Role of Natera: Natera, Inc. generated the personalized ctDNA assays and performed bioinformatic variant calling; Natera staff assisted with generation of Figs. 1B, 2, and 5. All clinical endpoints and outcome analyses were performed independently at MSK by institutional statisticians; patient management and outcome adjudication were conducted independent of the company.
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Romesser, P.B., Bercz, A., Alvarez, J. et al. Tumor-informed circulating tumor DNA stratifies recurrence risk and survival in anal squamous cell carcinoma. Nat Commun (2026). https://doi.org/10.1038/s41467-026-69984-y
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DOI: https://doi.org/10.1038/s41467-026-69984-y
