Tumor-informed circulating tumor DNA stratifies recurrence risk and survival in anal squamous cell carcinoma

Domain: chemistry
Published: 2026-02-26
Editorial outcome: minor_revision

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.

Introduction
Anal squamous cell carcinoma (ASCC) is a human papillomavirus (HPV)–associated malignancy for which definitive-intent (chemo)radiation (CRT) remains the standard treatment for non-metastatic disease.1-3 Although CRT often achieves long-term control and sphincter preservation, up to six months of serial evaluations may be required to assess treatment response.4 This prolonged interval may delay the detection of treatment failure, limit the window for effective salvage therapy, and contribute to patient anxiety and uncertainty in management. For patients with persistent or recurrent locoregional disease, salvage abdominoperineal resection (APR) remains the primary treatment, but it is associated with poor disease-specific and recurrence-free survival.5,6 These limitations underscore the need for early, reliable biomarkers to identify high-risk patients during or shortly after CRT, when timely therapeutic intervention may still improve outcomes.

Circulating tumor DNA (ctDNA) has emerged as a promising, minimally invasive blood-based biomarker that enables dynamic monitoring of disease burden through the detection of tumor-specific mutations in plasma.7-11 Prior studies in ASCC have predominantly focused on HPV-directed ctDNA assays,12-20 but these approaches may lack sensitivity in patients with HPV-negative (~5-10% of patients with ASCC).21,22 Tumor-informed ctDNA assays, customized to each patient’s unique mutational profile, may offer a more sensitive and broadly applicable alternative, although their utility in ASCC has not been well established yet.23,24

In this multicenter, real-world cohort study, we evaluated a commercially available, personalized, tumor-informed ctDNA assay for its prognostic and surveillance utility in patients with ASCC undergoing definitive-intent CRT. We hypothesized that ctDNA kinetics at pre-treatment, during-treatment, and end-of-treatment would correlate with clinical outcomes and, if validated, could inform risk-adapted, personalized treatment strategies.

Results
Patient, Tumor, and Treatment Characteristics
Of 88 consecutive patients with localized ASCC who underwent definitive-intent dose-painted intensity-modulated radiation therapy (DP-IMRT), 84 patients (95%) had sufficient tumor tissue from diagnostic biopsies for ctDNA testing and were included in the final cohort (Figure 1A). Whole-exome sequencing (WES) identified key mutations across this population (Figure 1B). Demographic and treatment characteristics are summarized in Table 1 and Supplementary Table 1. The median follow-up among survivors, measured from diagnosis, was 18 months (interquartile range [IQR], 11–26 months).

ctDNA Detection and Temporal Dynamics
Across all timepoints, 647 plasma specimens were analyzed for ctDNA testing using a personalized, tumor-informed multiplex polymerase chain reaction-next-generation sequencing (mPCR-NGS) assay (SignateraTM) (Figure 1C). Cell-free DNA (cfDNA) concentrations increased during CRT, but ctDNA detection remained unaffected (Figure 1D). At pre-treatment, 79% (61 of 77) of patients were ctDNA-positive, including 89% (24 of 27) with stage III disease. Pre-treatment ctDNA positivity was significantly associated with T3/T4 tumors (odds ratio [OR], 4.08; 95% confidence interval [CI], 1.01–27.6; P=0.049) and node-positive disease (OR, 8.33; 95% CI, 2.19–41.5; P=0.001) (Supplementary Table 2). As treatment progressed, ctDNA levels generally declined, though levels remained higher at each timepoint among patients with more advanced clinical stage, compared to stage I (Supplementary Figure 1 and Supplementary Table 3). Median ctDNA levels at pre-treatment, mid-treatment, and end-of-treatment were 1.02 MTM/mL (interquartile range (IQR), 0.17-25.43), 0 MTM/mL (IQR, 0-0.46), and 0 MTM/mL (IQR, 0-0). Of the end-of-treatment samples, 6 were drawn in the final week of CRT (−7 to -1 days), 7 on the final day of CRT (0 day), and 60 after CRT completion (+1 to +42 days) (Figure 1C).

Treatment Failure Events
The clinical courses of the cohort, grouped by baseline stage and outcome, are shown in Figure 2. Twelve patients (14%) experienced treatment failure (including death without progression) including seven locoregional failures, four distant failures, and one death (Supplementary Figure 2). Of the seven patients with locoregional failure, three had local-only failure, two had nodal-only failure, and two had combined local-nodal failure. Among patients with locoregional recurrence, three underwent salvage APR, two received nonoperative salvage with re-irradiation, and two were deemed medically ineligible for salvage therapy and was managed with best supportive care. Five deaths occurred in the post-CRT follow-up period. Four were attributed to disease progression including three from distant metastatic disease and one from locoregional recurrence as the patient died from complications of the recurrent tumor. At the last available disease assessment prior to death, all four had positive ctDNA. One additional patient died without evidence of disease progression due to complications of COVID-19 and Clostridioides difficile (C. difficile) colitis.

Among the 12 patients with treatment failure or death, 11 (92%) had at least one high-risk feature (tumor >4 cm and/or nodal involvement). All patients completed their prescribed course of CRT without dose reductions or treatment breaks. In a competing-risks analysis, absence of mitomycin at CRT and HPV status were not associated with increased hazard of locoregional recurrence (Supplementary Table 4). In all cases, ctDNA positivity preceded clinical or radiographic evidence of recurrence (Supplementary Figure 2). Notably, all patients who were ctDNA-negative at baseline remained recurrence-free during follow-up, suggesting this subgroup may represent particularly favorable biology.

Overall Survival, Progression-Free Survival, and Locoregional Failure
Pre-treatment ctDNA-negative patients had an estimated one-year overall survival (OS) of 100% (95% CI, 100-100%), compared to 97% (95% CI, 92-100%) for those with ctDNA-positive status (Figure 3A). Stratified by during-treatment ctDNA status, one-year OS was 100% (95% CI, 100-100%) for ctDNA-negative versus 93% (95 % CI, 84-100%) for ctDNA-positive patients (Figure 3B). At end-of-treatment, ctDNA-negative patients had a one-year OS of 96% (95% CI, 90-100%) versus 63% (95% CI, 32-100%) for ctDNA-positive patients (Figure 3C).

Similarly, one-year progression-free survival (PFS) was 100% (95% CI, 100-100%) for pre-treatment ctDNA-negative patients and 85% (95% CI, 76-96%) for ctDNA-positive patients (Figure 3D). During-treatment ctDNA-negative patients had a one-year PFS of 100% (95% CI, 100-100%), compared to 78% (95% CI, 62-98%) for ctDNA-positive patients (Figure 3E). End-of-treatment ctDNA-negative patients had a one-year PFS of 92% (95% CI, 84-100%) versus 44% (95% CI, 17-100%) for ctDNA-positive patients (Figure 3F).

Locoregional failure (LRF) at one year was 0% (95% CI, undefined) among ctDNA-negative patients versus 11% (95% CI, 4-23%) among ctDNA-positive patients at pre-treatment (Figure 3G). Pre-treatment quantitative ctDNA was not associated with LRF (HR 1.0, 95% CI 0.99-1.00). At during-treatment, one-year LRF was 0% (95% CI, undefined) versus 19% (95% CI, 5-38%) for ctDNA-negative and ctDNA-positive groups, respectively (Figure 3H). At end-of-treatment, the one-year LRF was 6% (95% CI, 2-16%) for ctDNA-negative versus 39% (95% CI, 2-79%) in ctDNA-positive patients (Figure 3I).

When stratified by the timing of initial ctDNA clearance, patients who were ctDNA-negative at pre-treatment or who cleared ctDNA during-treatment had a one-year OS of 100% (95% CI, 100–100%; Figure 4A), one-year PFS of 100% (95% CI, 100–100%; Figure 4B), and one-year LRF rate of 0% (95% CI, undefined; Figure 4C). Among patients who cleared ctDNA only at end-of-treatment, one-year OS was 89% (95% CI, 75–100% Figure 4A), PFS was 81% (95% CI, 65–100%; Figure 4B), and LRF was 15% (95% CI, 4–35%; Figure 4C). Patients who were ctDNA-positive at end-of-treatment had significantly worse outcomes with one-year OS of 63% (95% CI, 32–100%), PFS of 44% (95% CI, 17–100%), and LRF of 39% (95% CI, 2–79%) (log-rank P=0.01 for OS and P=2.4x10-6 for PFS; Gray’s P=0.001 for LRF). All patients who were ctDNA-negative at end-of-treatment achieved a clinical complete response.

ctDNA Surveillance Monitoring
Seven patients developed molecular recurrence during the surveillance period after initially achieving clearance (Figure 5). All these patients subsequently experienced confirmed relapse, five locoregional and two distant, with ctDNA re-positivity consistently preceding radiographic or clinical evidence of recurrence by a median of 2.4 months (range 1-8.7 months). Because ctDNA re-positivity triggered off-schedule ‘enhanced’ assessments, the 2.4-month median lead time in the surveillance subset likely underestimates the biological lead time relative to standard NCCN. Among all patients who were persistently ctDNA-negative during the surveillance period, no treatment failure events occurred.

Methods
Study Design and Participants
The institutional review and privacy boards of Memorial Sloan Kettering Cancer Center (MSK; IRB 16-370) and the University of South Florida (USF; IRB 004774) approved this cohort study with a waiver of informed consent. Patient confidentiality was maintained as required by the Health Insurance Portability and Accountability Act (HIPAA). Beginning in March 2021, adult patients (≥18 years) with biopsy-confirmed, non-metastatic ASCC were prospectively offered longitudinal ctDNA monitoring and all patients who provided informed consent to ctDNA testing were included. All patients were staged per the American Joint Committee on Cancer (AJCC), 8th edition30 and received curative-intent CRT. Data collection concluded on July 31, 2024. Data sharing was governed by a three-way agreement among MSK, USF, and Natera, Inc., to allow data to be shared freely across all participating institutions, with the final dataset maintained at MSK.

Personalized mPCR-NGS ctDNA assay
A clinically validated, personalized, tumor-informed 16-plex mPCR-NGS assay (SignateraTM, Natera, Inc.) was used for the detection and quantification of ctDNA in blood samples.31 Formalin-fixed, paraffin-embedded tumor tissue from surgical resection or biopsy samples and matched normal DNA extracted from a peripheral blood sample were processed for whole-exome sequencing to identify and track up to 16 patient-specific and tumor-specific somatic single-nucleotide variants (SNVs) in the associated patient’s plasma using a multiplex PCR-NGS approach. Cell-free DNA was extracted from patient plasma (median, 9.4 ml; range, 2.9–10.2 ml) at a given time point and was used to detect ctDNA. A plasma sample was classified as ctDNA-positive if two or more tumor-specific variants out of the 16 were detected above a predefined threshold. The predefined threshold is based on Natera’s proprietary variant calling method, wherein detecting at least two out of 16 variants ensures optimal analytical performance of the assay, with greater than 95% sensitivity at a mean variant allele frequency of 0.01% and 99.7% specificity.32 ctDNA concentration was reported as mean tumor molecules per milliliter (MTM/mL) of plasma. Testing cadence was at the discretion of the treating physician, with results available in real time. Lead time was defined as the interval between the first ctDNA re-positivity after a documented negative result and the date of first clinical or radiographic evidence of recurrence.

Treatment and Response Assessment
All patients received curative-intent CRT, typically with DP-IMRT. Radiation dose was adapted to tumor size: <2 cm, 50 Gy; 2–4 cm, 54 Gy; >4 cm, 58 Gy, all delivered in 2-Gy fractions.6 Most patients received concurrent fluoropyrimidine-based chemotherapy, with or without mitomycin, unless clinically contraindicated.

Treatment response was assessed 12–16 weeks after CRT using physical examination (including digital rectal and inguinal lymph node exams), anoproctoscopy, and cross-sectional imaging (MRI, CT, or PET-CT). Clinical complete response (cCR) was defined as no detectable tumor locally, regionally, or distantly. For patients with an incomplete but improving primary tumor response, short-interval re-evaluation (within 4–8 weeks) was permitted. Recurrence was defined as local, regional, or distant disease. Locoregional failure was defined as recurrence at the primary site or in pelvic/inguinal regional nodes, with ‘combined local–nodal failure’ reserved for concurrent recurrence at both sites. Salvage management for locoregional recurrence was categorized as operative salvage (APR), nonoperative salvage (re-irradiation and/or systemic therapy), or best supportive care for patients medically ineligible for salvage therapy.

Phlebotomy Timeline
Peripheral blood was collected at physician-defined intervals throughout the treatment course (Supplementary Figure 3). Pre-treatment samples were obtained at any point before treatment initiation or within five days after starting CRT. During-treatment samples were collected more than five days after CRT initiation but at least seven days before its completion. End-of-treatment samples were drawn from seven days before through 42 days after the end-of-CRT. Samples collected more than 42 days after CRT completion were categorized as surveillance timepoints. Patients who re-converted from negative to positive ctDNA status during surveillance underwent ‘enhanced surveillance’, including directed clinical examination (digital rectal examination and, when appropriate, flexible sigmoidoscopy/anoscopy) and expedited cross-sectional imaging (MR pelvis, CT chest/abdomen/pelvis, and/or PET-CT).

Study Endpoints
The primary endpoint was OS, defined as the time from ctDNA assessment to death from any cause or to last follow-up. Secondary endpoints included progression-free survival and the cumulative incidence of locoregional failure. PFS was defined as the time from ctDNA assessment to the first occurrence of local or regional failure, distant metastasis, or death. LRF was defined as persistent, progressive, or recurrent disease in the primary tumor or regional lymph nodes, with death and distant recurrence treated as competing-risks.

Statistical Analyses
Baseline characteristics were summarized using medians and interquartile ranges (IQR) for continuous variables, and frequencies with percentages for categorical variables. Univariate logistic regression was used to evaluate predictors of pre-treatment ctDNA positivity. Kaplan-Meier estimates were used to evaluate OS and PFS from the date of end-of-treatment ctDNA assessment, stratifying patients by time of clearance. Additional timepoint-specific Kaplan-Meier analyses were conducted for OS and PFS according to ctDNA status at each sampling point, measured from the corresponding ctDNA collection date. Survival differences were compared using log-rank tests. LRF was analyzed at the same timepoints using competing-risk regression, with cumulative incidence curves by Gray’s test.

Patients without a ctDNA result at a given time point were excluded from time point-specific survival analyses. If multiple samples existed in a given phase (e.g., during-treatment), the most recent test was selected. In surveillance, if all ctDNA results were negative, the most recent was used; if any were ctDNA-positive, the first positive test was selected. Analyses were performed in R (v4.4.0) using tidyverse (v2.0.0), gtsummary (v2.0.0), ggsurvfit (v1.1.0), ggalluvial (v0.12.5), and ggpubr (v0.6.0).

Peer review rounds
[{"round": 1, "reviewer_comments": {"reviewer_1": "To the authors,\nThe investigators have created a two-institution evaluation of 84 locally advanced anal CA patients and have obtained a\nbaseline tumor informed ctDNA and a post treatment post ctDNA and compared outcomes.\nThe authors concluded that a positive tumor informed ctDNA post treatment was associated with outcomes and should be\nconsidered as part of the standard evaluation for response. The primary endpoint was OS; secondary endpoints were PFS\nand LRF.\nComments:\n• Please revise the statement: “….determining treatment response often requires up to six months of clinical and\nradiographic follow-up.” I would suggest a revision of the verbiage “up to 6M may be required for serial evaluations to\ndetermine optimal outcomes for sphincter preservation.”\n• Was there a correlation of pretreatment ctDNA with AJCC staging?\n• Of the 12 patients with recurrence, did they exhibit high-risk features? Tumor > 4 cm and/or lymph node positivity? Were\nthey compliant with their radiation? Did they have any dose reductions or delays?\n• Was there any difference in outcomes for patients that were HPV negative?\n• Was there any difference for the 7% of pts that did not receive MMC?\n• I would recommend removing the one patient with the treatment that is unknown\n• I think there is a mistake in your table re: HPV. It states 100% were positive or negative but 29 are unknown?\n• 5-Fluorouracil is misspelled in the table C.\n• Is the evaluation mid-treatment ctDNA necessary?\n• The author states the use of ctDNA will allow “earlier escalation for high-risk patients, and de-intensification for molecular\nresponders”…. Are you suggesting that we should revise our surveillance or consider an APR sooner? I am not sure that a\npositive ctDNA would convince patients to have their APR sooner if there is no evidence clinically for residual disease?", "reviewer_2": "Reviewer Comments to the Authors\nThis manuscript addresses an important and practical topic in anal squamous cell carcinoma (ASCC) management,\nevaluating a tumor informed circulating tumor DNA (ctDNA) assay in a real world cohort. This study is a valuable\ncontribution, presenting real world data on tumor informed ctDNA testing in ASCC. Its strengths include prospective data\ncollection, excellent assay performance, and clinical relevance. To strengthen scientific rigor and interpretability, revisions\nshould address assay quantification, sampling timing, subgroup analyses, contextual comparison with HPV based assays,\nand careful framing of conclusions.\nComments\n1. Define the Negative Baseline Group and High Burden Misses\nPlease provide the characteristics of the ctDNA negative patients at baseline—particularly the 11% with high burden/stage\nIII disease. Were these predominantly HPV negative or did they have lower mutational burden? Can the authors clarify the\nmolecular and clinical details of this subgroup (e.g., mutation profiles, HPV status, stage distribution).\n2. Assay Description and Quantitative Values\nThe manuscript currently provides only binary ctDNA status. Please include:\no The commercial name of the assay (e.g., Signatera™, Natera Inc.) in the Abstract and Methods.\no The reason for calling ctDNA positive (e.g., number of SNV targets).\no Include quantitative values such as median levels at baseline and during therapy in the text (only in Fig 2E currently), and\nwhether higher or lower ctDNA quantity, or rate of decline, correlates with outcomes. If there is a correlation with pre-\ntreatment level and outcome, it could be included in the multivariate analysis.\n3. Timing of Sampling: Refinements Suggested\nThe definitions of baseline, mid treatment, and post treatment sampling are broad and could introduce bias.\no The time windows chosen allow for pre-treatment to occur 5 days after starting CRT, while post-treatment can be 7 days\nbefore completion. The definitions used raise the question whether the dates were chosen post-hoc to influence the results.\nDo the results change with the more commonly accepted definitions of pre and post treatment? I would suggest changing the\ndefinition. Otherwise, consider renaming “mid-treatment” to “during treatment” and “post treatment” to “end of treatment” for\nclarity.\no Specify how many patients had samples taken in the final week of treatment versus genuinely post treatment.\n4. Sample Counts by Time Point\nPlease provide a clear breakdown of the number of patients with ctDNA results at each time point (baseline, during-\ntreatment, end-of-treatment, surveillance), including missing data and how these were handled in survival analyses.\n5. Interpretation of Clearance and Clinical Applicability\nWhile early ctDNA clearance is promising, caution is needed given the modest cohort size (84 patients), low event count (12\nfailures), and limited follow up (~18 months). Statements like “lays the foundation for integration into care” should be\ntempered to reflect a hypothesis-generating study awaiting prospective validation.\n6. Comparison with HPV ctDNA Assays\nPlease include a discussion comparing tumor informed ctDNA with HPV based ctDNA testing. For example, the TTMV HPV\nDNA assay (Kabarriti et al., Cancers, 2025) demonstrated per patient surveillance sensitivity of 82.8%, specificity of 98.4%,\nPPV of 96.0%, and NPV of 92.5%, with valuable quantitative ranges and lead time data pubmed.ncbi.nlm.nih.gov+1. How\nmight your approach compare in terms of sensitivity, specificity, and quantification, including in HPV-negative disease? It\nwould be ideal to provide outcomes for the HPV negative patients (approximately n=6). How did the assay perform at\nbaseline and during surveillance in this subgroup? This is important to distinguish your tumor informed approach from viral\ntargeted tests like NavRx.\n7. ctDNA Detection of Recurrence During Surveillance\nConfirm whether ctDNA positivity preceded all recurrences, including those detected during routine surveillance. Were there\nany relapses not captured by ctDNA testing? If so, why might that have occurred? In the small cohort of patients with\nnegative pre-treatment ctDNA, was the surveillance testing useful?\n8. False Negatives After Clearance\nPatients who cleared ctDNA during treatment but later recurred suggest potential low disease burden below detection\nthreshold. Please discuss this limitation and whether alternative threshold levels (e.g., MTM/mL at a given time point) might\nenhance prediction of durable remission. This area is a potential area of harm for clinicians, in that there may be a false\nsense of assurance for a favorable outcome with a mid-treatment clearance, that could result in inappropriate treatment de-\nescalation.\n9. Testing Cadence and Clinical Influence\nSince testing frequency was at physician discretion, clarify how this may have impacted results. Did ctDNA results ever\ninfluence clinical decision making during the study? If so, please describe.\n10. Avoid Overinterpretation\nRephrase any language suggesting that the assay is ready for routine clinical integration. Emphasize that findings support\nthe design of prospective trials rather than immediate practice changes.\n11. Conflict of Interest and Industry Roles\nAcknowledge in the relevant sections that six co authors are employees of Natera and clarify the company's role in assay\ndevelopment, data collection, statistical analysis, and manuscript drafting.\n12. Interpretation of cfDNA Plot\nPlease add a brief explanation for the cfDNA concentration plot (e.g., in Figure 2a). Specifically, note that although cfDNA\nlevels rise during treatment, ctDNA detection remains specific—highlighting assay robustness.", "reviewer_3": "I co-reviewed this manuscript with one of the reviewers who provided the listed reports. This is part of the Nature\nCommunications initiative to facilitate training in peer review and to provide appropriate recognition for Early Career\nResearchers who co-review manuscripts."}, "author_response": "Authors response à Thank you for this suggestion. We have revised the sentence as\nrecommended to clarify that up to six months of serial evaluations may be required to\ndetermine optimal outcomes for sphincter preservation.\nAuthors response àThank you for this question. We have updated the Results (Fig. 2c–d) to\nclarify this point. Pretreatment ctDNA positivity was significantly associated with higher\nAJCC T and N stage. Specifically, positivity was enriched in patients with T3/T4 tumors (OR\n4.08; 95% CI, 1.01–27.6; P=0.049) and node-positive disease (OR 8.33; 95% CI, 2.19–41.5;\nP=0.001). By AJCC stage, pretreatment ctDNA positivity was 30% in Stage I, 85% in Stage II,\nand 89% in Stage III.\nAuthors response à Thank you for this question. Among the 12 patients who recurred, 10\n(83%) had at least one high-risk feature (tumor >4 cm and/or nodal involvement). All\npatients completed their prescribed course of radiotherapy without gross non-\ncompliance; no patients experienced major dose reductions or treatment interruptions.\nWe have added this detail to the Results section.\nAuthors response à HPV status was available for 55 patients, of whom 48 (87%) were HPV-\npositive and 7 (13%) were HPV-negative HPV status is not associated with locoregional\nfailure when accounting for distant recurrence and death as competing events, however\nour limited sample size of HPV negative patients and locoregional failures limits our\ninterpretation.\nAuthors response à Thank you for this question. We did not observe any association\nbetween patients that did not receive MMC and locoregional failure when accounting for\ndistant recurrence and death as competing events. We have updated the results to\nspecifically state this:\nIn an exploratory competing-risks analysis, absence of mitomycin at\nchemoradiation was not associated with locoregional failure.\nGiven that the number of patients who did not receive MMC was small, further studies may\nbe needed to validate these findings.\nAuthors response à Thank you for this suggestion. For the patient in question, we have\ncomplete details of the radiation course and have spoken with the medical oncologist who\nconfirmed that this patient was treated with radiation alone. We have updated Figure 1c to\nappropriately reflect this. As this patient was treated with definitive-intent radiation and\ngiven this patient contributes evaluable outcome data, we have retained this patient in the\ncohort.\nAuthors response à Thank you for pointing out this discrepancy. HPV status was available\nfor 55 patients, of whom 48 were positive and 7 negative. An additional 29 patients were\nnot tested and are therefore listed as “unknown.” We have revised the table to reflect all\n84 patients in the denominator, with a separate “unknown” category for clarity.\nAuthors response à Thank you. This has been corrected.\nAuthors response à We appreciate this insightful comment. In our study, mid-treatment\nctDNA testing identified a subgroup of patients who achieved early molecular clearance,\nall of whom had excellent outcomes with no observed events for OS, PFS, or LRF.\nThis\nsuggests that mid-treatment ctDNA may provide an early readout of treatment sensitivity\nand help identify patients most likely to benefit from standard, sphincter-preserving\nmanagement without intensification. While the end-of-treatment timepoint showed the\nstrongest prognostic separation, the mid-treatment findings add complementary\ninformation by highlighting a subset of “early molecular responders.” We agree that\nvalidation in larger cohorts is needed, but these results support the potential value of mid-\ntreatment ctDNA in informing future eiorts to personalize therapy.\nAuthors response à We appreciate this important question. Our intention is not to suggest\nthat ctDNA positivity alone should prompt abdominoperineal resection (APR) in the\nabsence of clinical or radiographic evidence of residual disease. Rather, we envision ctDNA\nas a complementary biomarker that could guide the intensity and timing of\nsurveillance and help identify patients most appropriate for enrollment in clinical trials of\nadjuvant or escalation strategies. For example, ctDNA-positive patients at the end of\ntreatment may warrant closer follow-up or trial enrollment, whereas patients achieving\nearly ctDNA clearance could eventually be considered for de-intensified surveillance once\nvalidated in larger cohorts. Thus, our data support the concept of ctDNA-informed risk\nstratification, but we agree that surgical decision-making should remain anchored in\nestablished clinical and radiographic assessments.\nAuthors response à Thank you for this comment. Detailed patient-level data are provided\nin Fig. 1b. In addition, we have expanded Fig. 1c to include adjacent columns stratifying\npatients by baseline ctDNA status (“Baseline Positive” vs. “Baseline Negative”), allowing\ndirect comparison of clinical stage, HPV status, and mutational profiles across groups.\nNotably, while ~11% of patients with stage III disease were ctDNA-negative at baseline, this\nsubgroup did not show enrichment for HPV-negative status or distinct mutational features.\nThese findings suggest that baseline ctDNA negativity, even in higher-stage disease, may\nreflect biological diierences in tumor shedding rather than inconsistencies in clinical or\nmolecular data.\nAuthors response à We thank the reviewer for these suggestions. We have now added the\ncommercial name (Signatera™, Natera Inc.) to the Abstract. As described in the Methods,\nctDNA positivity was defined as ≥2 SNVs detected above assay-specific thresholds. We\nexpanded the methods section with additional details on the assay. The description is\naligned with other studies published with Signatrea in Nature Medicine. We have also\nincluded quantitative ctDNA values in the Results (median baseline, during-treatment, and\nend-of-treatment levels; Fig. 2e) and report exploratory analyses of whether baseline\nburden or rate of decline correlated with outcomes.\nAuthors response à Thank you for this clarifying question. The detailed methodology of\nSignatera assay was previously published in JAMA Oncology (Reinert et al. 2019) and the\nanalytical validation was published in Clinical Cancer Research (Coombes et al. 2019). We\nhave now provided both these references in the methods and have further expanded the\nmethodology to provide a brief explanation of the assay as follows:\nA clinically validated, personalized, tumor-informed 16-plex polymerase chain\nreaction (PCR) next-generation sequencing (NGS) assay (SignateraTM, Natera, Inc.)\nwas used for the detection and quantification of ctDNA in blood samples.27 In brief,\nformalin-fixed, paraffin-embedded tumor tissue from surgical resection or biopsy\nsamples and matched normal DNA extracted from a peripheral blood sample were\nprocessed for whole-exome sequencing to identify and track up to 16 patient-\nspecific and tumor-specific somatic single-nucleotide variants (SNVs) in the\nassociated patient’s plasma using a multiplex PCR-based NGS approach. Cell-free\nDNA was extracted from patient plasma (median, 9.4 ml; range, 2.9–10.2 ml) at a\ngiven time point and was used to detect ctDNA. A plasma sample was classified as\nctDNA-positive if two or more tumor-specific SNVs out of the 16 were detected\nabove a pre-defined threshold. The predefined threshold is based on Natera’s\nproprietary variant calling method, wherein detecting at least two out of 16 variants\nensures optimal analytical performance of the assay, with greater than 95%\nsensitivity at a mean variant allele frequency of 0.01% and 99.7% specificity28.\nctDNA concentration was reported as mean tumor molecules per milliliter\n(MTM/ml) of plasma. Testing cadence was at the discretion of the treating\nphysician, with results available in real time.\nReferences:\n• Reinert, T. et al. Analysis of plasma cell-free DNA by ultradeep sequencing in\npatients with stages I to III colorectal cancer. JAMA Oncol. 5, 1124–1131 (2019).\n• Coombes, R. C. et al. Personalized detection of circulating tumor DNA antedates\nbreast cancer metastatic recurrence. Clin. Cancer Res. 25, 4255–4263 (2019).\nAuthors response à Thank you for this suggestion. In the revised manuscript, we now\ninclude quantitative ctDNA values at baseline and during therapy in the text (in addition to\nFig. 2e). Median ctDNA levels and their ranges are reported to provide context for assay\nperformance. While exploratory analyses of rate of decline and kinetics are of great\ninterest, these were beyond the scope of the present study and are the subject of ongoing,\nmore detailed work. In this analysis, we focused on ctDNA status at clinically relevant\ntimepoints (baseline, during, and end-of-treatment), which provided robust prognostic\ndiscrimination and are most directly aligned with potential clinical application.\nAdditionally, pre-treatment ctDNA levels were not associated with locoregional failure\nwhen accounting for distant recurrence and death as competing events.\nAuthors response à We appreciate this important point. The sampling windows were\ndefined a priori based on pragmatic clinical and logistical considerations, with the goal of\nmaximizing the number of blood draws available per patient at each time point. These\ndefinitions were first presented publicly in an abstract and oral presentation at the 2023\nASCO Gastrointestinal Cancers Symposium (J Clin Oncol 41, 1–1, 2023), well before\nclinical outcome data were available. Thus, the windows could not have been selected\npost hoc to optimize associations with outcomes. As the dataset matured and clinical\nevents accumulated, we deliberately maintained these predefined intervals to preserve\nmethodological consistency.\nThat said, we recognize that the original terminology (“mid-treatment” and “post-\ntreatment”) may have implied greater flexibility than intended. To address this, we have\nrevised the manuscript to use “during treatment” and “end-of-treatment” as suggested.\nWe also now provide the number of patients whose samples were collected in the final\nweek of CRT versus after CRT completion, to clarify the temporal distribution of draws.\nAuthors response à Thank you for this important request. Detailed timing of all 647 cfDNA\ndraws is provided in Fig. 2a, where samples are color-coded by draw category. In the\nrevised manuscript, we now specify the number of patients with samples collected in the\nfinal week of CRT (–7 to 0 days relative to treatment completion) versus those collected\ngenuinely post-treatment (0 to +42 days). Specifically, 6 patients had draws within –7 to 0\ndays, while 67 patients had draws within 0 to +42 days after CRT. As noted, these sampling\nwindows were defined a priori and applied consistently throughout the study to maximize\ndata availability while maintaining methodological consistency. We updated the results to\nclearly define the timing of the end-of-treatment samples:\nOf the end-of-treatment samples, 6 patients were drawn in the final week of CRT (−7\nto -1 days), 7 patients were draw on the final day of CRT (0 day), and 60 patients were\ndrawn after CRT completion (+1 to +42 days) (Fig. 2a).\nAuthors response à We have added a Cohort flow diagram (Fig. 1a) that specifies sample\ncounts at baseline, during treatment, end-of-treatment, and surveillance, including\nmissing data. For survival analyses, only patients with samples within the specified\nwindows were included; those without data at a given time point were excluded from that\nspecific analysis.\nTime Point # Patients with Data # Patients Missing Data\nAvailable (No Test in that Time Point)\nBaseline 77 7\nDuring treatment 62 22\nEnd-of-treatment 73 11\nSurveillance 77 7\nIn all survival analyses, the 4 patients that failed the Natera tests were excluded.\nPatients with missing data were excluded from analyses where “time 0” is the date of that\ntimepoint. For example, the 7 patients missing baseline ctDNA draws were excluded from\nany analysis where time started at baseline draw (for OS, PFS, and locoregional failure).\nFor survival analyses that stratify by “time of clearance” -- there are 10 patients that were\nexcluded for other reasons:\n1. They were detectable at baseline but did not have a during or end-of-treatment test.\n2. They were detectable at baseline and during treatment but did not have an end-of-\ntreatment test.\n3. They didn’t have a baseline, during, or end-of-treatment test (only Surveillance)\nWe explain how we handle multiple tests per timepoint in the manuscript (in Methods:\nStatistical Analyses) -- “If multiple samples existed in a given phase (e.g., during mid-\ntreatment), the most recent test was selected. In surveillance, if all ctDNA results were\nnegative, the most recent was used; if any were ctDNA-positive, the first positive test was\nselected.”\nAuthors response à We agree with the reviewer that these findings must be interpreted\nwith caution given the modest sample size, low event count, and relatively short follow-up.\nWe have revised the Discussion to frame early clearance as a hypothesis-generating\nobservation that supports future validation, rather than suggesting immediate clinical\napplication.\nAuthors response à We thank the reviewer for this important suggestion and have added\na dedicated section to the Discussion comparing tumor-informed and HPV-based assays.\nViral-based assays such as TTMV-HPV DNA (NavDx®) have shown strong performance in\nHPV-positive anal cancer, with per-patient surveillance sensitivity of 82.8%, specificity of\n98.4%, PPV of 96.0%, and NPV of 92.5%, and a median lead time of 59 days before clinical\nrecurrence. These results highlight the value of viral assays, particularly given their\nsimplicity and lack of requirement for tumor tissue.\nHowever, viral-targeted approaches are not applicable to HPV-negative disease, which\naccounts for approximately 10–15% of ASCC. In contrast, tumor-informed ctDNA assays\nare tailored to each patient’s mutational profile, enabling detection across both HPV-\npositive and HPV-negative disease. In our cohort, outcomes were similar for HPV-positive\n(7/48, 14.5% recurrence) and HPV-negative patients (1/7, 14.3% recurrence), underscoring\nthe applicability of tumor-informed monitoring across subgroups despite the low\nmutational burden of ASCC. Importantly, all patients who were ctDNA-negative at baseline\nremained recurrence-free, regardless of HPV status.\nAs we have recently emphasized in an editorial, next-generation sequencing–based\nctHPVDNA assays in oropharyngeal cancer consistently demonstrate higher sensitivity\ncompared with ddPCR-based methods, including detection in earliest-stage disease and\nlonger lead times for recurrence. These observations suggest that both viral-based and\ntumor-informed approaches have complementary strengths. Viral assays may offer\nscalability in HPV-positive populations, whereas tumor-informed testing provides\nuniversal applicability, including in HPV-negative disease, and may allow for integration\nwith non-viral biomarkers.\nAuthors response à We confirm that ctDNA positivity preceded all instances of clinical or\nradiographic recurrence, including those first detected on routine surveillance. Importantly,\nno relapses were missed by ctDNA testing. The median lead time between ctDNA\ndetection and clinical/radiographic recurrence was 5.7 months (range, 1.3–13.5 months),\nunderscoring the potential value of ctDNA as an early signal of relapse. We also note that\npatients who converted from undetectable to detectable ctDNA during surveillance were\nplaced on enhanced follow-up, which may have shortened the observed lead time\ncompared with what might have been seen under standard surveillance protocols. Finally,\nall patients with negative baseline ctDNA remained recurrence-free during follow-up,\nsuggesting this subgroup may represent particularly favorable biology. We have clarified\nthese points in the Results section\nAuthors response à We agree with the reviewer that recurrence after apparent clearance\nlikely reflects disease burden below the assay’s detection threshold, underscoring the\nneed for cautious interpretation of isolated timepoints. In our study, the patients with the\nmost favorable outcomes were those who not only cleared ctDNA, but also remained\nctDNA-negative on subsequent surveillance draws. By contrast, patients who recurred\ntypically demonstrated re-emergence of ctDNA positivity prior to clinical or radiographic\nrelapse. These findings suggest that serial monitoring provides important complementary\ninformation, whereas relying on clearance at a single timepoint may be insuiicient.\nImportantly, this distinction strengthens the rationale for continued surveillance rather\nthan treatment de-escalation based on a single negative result. While emerging\ntechnologies (e.g., WGS-based assays or alternative thresholds) may further improve\nsensitivity, our results support the clinical value of serial monitoring with current assays.\nAuthors response à We appreciate this helpful comment. While testing cadence was\nultimately at physician discretion, it was recommended that blood draws be obtained at\npredefined intervals: pre-treatment, during-treatment, end-of-treatment, 1 month post-\ntreatment, and at 3-month intervals thereafter. As this was a multi-institutional, real-world\ncohort, there was expected variability in adherence to these recommended draws.\nAll patients were otherwise managed according to institutional standards. ctDNA results\nwere returned in real time and, in select cases, did prompt additional evaluation. For\nexample, in one patient, molecular recurrence after nearly three years of sustained\nclearance triggered early imaging, which identified a solitary resectable liver metastasis;\nthis patient remains disease-free at last follow up.\nAuthors response à We thank the reviewer for this comment. We have revised the\nDiscussion to temper language that might imply readiness for routine clinical integration.\nInstead, we now explicitly frame our findings as hypothesis-generating and emphasize that\nthey require validation in larger, prospective cohorts before influencing clinical practice.\nAuthors response à We now explicitly acknowledge that six co-authors are employees and\nequity holders of Natera, Inc. In addition to ensure transparency, we have updated the\nmethods and disclosure statement to be more explicit.\nMethods:\nA three-way data use agreement was established to allow data to be shared freely\nacross all participating institutions, with the final dataset maintained at Memorial\nSloan Kettering Cancer Center (MSK).\nDisclosure:\nRole of Natera: Natera, Inc. generated the personalized ctDNA assays and\nperformed bioinformatic variant calling; Natera stai assisted with generation of\nFigures 1b, 2a–b, 3, and 6. All clinical endpoints and outcome analyses were\nperformed independently at MSK by institutional statisticians; patient management\nand outcome adjudication were conducted independent of the company.\nAuthors response à We clarified this point in the Results, and for clarity, added two\nsentences to the Figure 2 legend."}, {"round": 2, "reviewer_comments": {"reviewer_1": "To the authors,\nThank you for your revised manuscript. However, I still some concerns about the content:\n1. I would revise this sentence: \"These data support ctDNA as a dynamic, treatment-responsive biomarker that warrants\nprospective validation as the basis for risk-adapted surveillance and adjuvant treatment in ASCC\". I would recommend\nreduced emphasis on \"support\" given the small sample size with short-term follow up.\nI would suggest: \"These data support the consideration of ctDNA as a dynamic, treatment-responsive biomarker that\nwarrants prospective validation as the basis for risk-adapted surveillance and adjuvant treatment in ASCC\"\n2.The authors mention more than once that this ctDNA assay may be more beneficial than a HPV ctDNA assay since the\nassay would not be beneficial in HPV negative patients. However, the incidence of HPV (-) tumors is rare <5% and should\nbe noted.\n3. 7 pts had ctDNA (+) at end of treatment or surveillance with 5 relapse—five locoregional and two distant—with ctDNA re-\npositivity consistently preceding radiographic or clinical evidence of recurrence by a median of 2.1 months. I cannot find any\nmention of salvage APR surgery for those that had evidence of local recurrence. Were there any patients that did not\nundergo salvage APR?\n4. Pls provide more information re: the one death\n5. Pls elaborate: For patients who re-converted from negative to positive during surveillance, \"enhanced\" clinical and/ or\nradiographic assessments were often initiated, likely shortening the observed lead time relative to standard NCCN\nsurveillance. What does \"enhanced\" mean and did it make a difference to shorten the diagnostic imaging time.\n6. There is also no mention of the psychosocial stress of testing for MRD and the implications.", "reviewer_2": "The authors have thoroughly addressed each of the points raised in the prior review. This is well done and suitable for\npublication in my opinion."}, "author_response": "Authors response  Thank you for this comment. We agree and adopted the reviewer’s phrasing in both the\nAbstract and the Discussion. The relevant sentence now reads: “These findings support the consideration of ctDNA\nas a dynamic, treatment-responsive biomarker that warrants prospective validation as the basis for risk-adapted\nsurveillance and adjuvant treatment in ASCC.”\nAuthors response  Thank you for this comment. We appreciate this important nuance. We now state explicitly that\nHPV-negative ASCC is rare (<5-10%) and clarify that our aim is complementarity, not replacement: tumor-informed\nctDNA can serve the small HPV-negative subset and provide an orthogonal signal alongside ctHPV DNA platforms in\nHPV-positive disease. We cite recent reports on ctHPV DNA performance and briefly discuss strengths/limitations to\npresent a balanced view.\nAuthors response  Thank you for this comment. The results have been updated to reflect salvage treatment for all\npatients who experienced treatment failure.\nAuthors response  Thank you for this comment. The results have been updated with “Five deaths occurred in the\npost-CRT follow-up period. Four were attributed to disease progression including three from distant metastatic\ndisease and one from locoregional recurrence as the patient died from complications of the recurrent tumor. At the\nlast available disease assessment prior to death, all four had positive ctDNA. One additional patient died without\nevidence of disease progression due to complications of COVID-19 and Clostridioides difficile (C. difficile) colitis.”\nAuthors response  Thank you for this comment. We revised Methods (Phlebotomy Timeline) to define “enhanced”\nas off-schedule clinical evaluation (digital rectal exam ± anoscopy) and expedited imaging (MR pelvis, CT\nchest/abdomen/pelvis, and/or PET-CT) prompted by ctDNA re-positivity. In the Discussion, we note that because\nctDNA re-positivity triggered off-schedule evaluation, the observed 2.4-month median lead time is likely conservative\nrelative to standard NCCN surveillance intervals.\nAuthors response  Thank you for this comment. We added a Limitations statement: we did not prospectively collect\nvalidated PROs on psychosocial impact; however, all participants consented with the option to discontinue testing, no\npatient requested discontinuation, and no testing-related distress was reported in routine encounters. We agree this\nis important and will plan to incorporate validated PRO measures prospectively.\nAuthors response  We sincerely thank the Reviewer for this supportive assessment. We also performed minor\ncopy-edits for clarity and consistency across the Abstract, Methods, Results, and figure legends.\nWe appreciate the editors’ and reviewers’ constructive feedback and believe the revisions improve the clarity,\nbalance, and clinical relevance of the manuscript. We are happy to address any remaining points."}]

Decision letter