Tumor-Informed ctDNA Stratifies Risk in Anal Squamous Cell Carcinoma

Key Takeaways

• End-of-treatment ctDNA positivity was associated with worse 1-year overall survival, progression-free survival, and locoregional failure, including 96% versus 63% overall survival and 92% versus 44% progression-free survival for ctDNA-negative versus ctDNA-positive patients.
• Patients who were ctDNA-negative at baseline or cleared ctDNA during treatment had 100% locoregional failure-free survival.
• Surveillance ctDNA re-positivity preceded confirmed relapse in every observed case.

In a real-world two-center cohort of adults with non-metastatic anal squamous cell carcinoma treated with curative-intent chemoradiation, serial tumor-informed ctDNA testing in the Nature Communications article identified seven surveillance molecular recurrences, all of which preceded confirmed clinical or radiographic relapse by a median of 2.4 months. Personalized longitudinal sampling spanned treatment and follow-up, and ctDNA patterns shifted across pretreatment, during-treatment, end-of-treatment, and surveillance phases. In this cohort, ctDNA was associated with both prognosis and relapse surveillance.

The study included 84 of 88 consecutive patients who had adequate tumor tissue for testing, and all had biopsy-confirmed non-metastatic anal squamous cell carcinoma treated with definitive-intent chemoradiation. Investigators used a personalized tumor-informed 16-plex mPCR-NGS Signatera assay built from whole-exome sequencing of formalin-fixed tumor tissue and matched normal blood. ctDNA was reported as mean tumor molecules per milliliter, and positivity required at least 2 of 16 patient-specific variants above the predefined threshold. Across 647 plasma specimens, sampling spanned pretreatment, during-treatment, end-of-treatment, and surveillance windows, allowing phase-specific comparisons.

Before treatment, ctDNA was detected in 79% of tested patients, including 89% of those with stage III disease. Pretreatment positivity was associated with bulkier primary tumors and nodal involvement, with reported odds ratios of 4.08 for T3 or T4 tumors and 8.33 for node-positive disease. During chemoradiation, patients who were ctDNA-negative had 1-year progression-free survival of 100% compared with 78% for ctDNA-positive patients. Patients who were ctDNA-negative at baseline or cleared ctDNA during treatment also had 100% locoregional failure-free survival.

Treatment completion provided the clearest prognostic separation in the cohort. At end of treatment, ctDNA-negative patients had 1-year overall survival of 96% versus 63% for ctDNA-positive patients. One-year progression-free survival was 92% versus 44%, and locoregional failure was 6% versus 39%, respectively. ctDNA positivity at treatment completion was associated with the least favorable short-term outcomes in this cohort.

During surveillance, physician-defined testing cadence allowed real-time review of results, and re-positivity led to enhanced surveillance with directed examination and expedited imaging. That workflow may have shortened the interval to confirmed relapse, making the observed lead time a conservative estimate. Median follow-up among survivors was 18 months, with an interquartile range of 11 to 26 months. No participant requested discontinuation or reported testing-related distress during clinical encounters, although patient-reported outcomes were not formally assessed. The investigators noted modest cohort size, few events, limited follow-up, and small subgroup analyses, and called for prospective validation before broader routine adoption or de-escalation inferences.