Dynamic ctDNA Assessment in Cervical and Anal Canal Tumors: Optimizing Follow-up and Clinical Outcomes (ANA)

Dynamic Assessment of ctDNA in Patients With Cervical and Anal Canal Tumors to Optimize Follow-up and Clinical Outcomes in the Brazilian Unified Health System (SUS)

After definitive radiotherapy (RT) treatment (with or without chemotherapy), cervical and anal canal neoplasms frequently exhibit disease persistence or recurrence. Due to the local inflammatory process post-treatment, response assessment by imaging (current gold standard) is limited, often necessitating multiple follow-ups and repeated invasive biopsies. Conventional follow-up is complex and costly, requiring equipment from secondary and tertiary services, trained radiologists, and patient exposure to radiation and contrast.

In this context of human papillomavirus(HPV)-related neoplasms, recent studies have demonstrated the role of ctDNA (circulating tumor DNA) in assessing the risk of recurrence or disease progression, providing a rationale for using the tool in two fronts:

• Optimizing follow-up based on serial monitoring of ctDNA;
• Selecting patients with positive ctDNA after RT, who are at high risk of recurrence, for treatment intensification.

Monitoring with ctDNA as a standalone follow-up tool in cases evolving with negative ctDNA after RT has the potential to replace imaging exams, being a minimally invasive test performed on a peripheral blood sample. Currently, ctDNA testing has expensive methodologies not available in the Unified Health System (SUS). This project aims to develop a methodology for ctDNA evaluation focused on HPV ctDNA research that is low-cost and executable in SUS, as well to assess the accuracy of this test in the population with HPV-related tumors.

Additionally, we will evaluate whether the early introduction of immunotherapy in patients with positive ctDNA after definitive treatment can increase cure rates. Immunotherapy already has a well-defined role in the treatment of metastatic HPV-related neoplasms. Recently, the use of anti-programmed death-1 (anti-PD1) has also shown benefits in patients with locally advanced cervical cancer with a high risk of recurrence who are candidates for chemoradiotherapy (CRT). Therefore, its use focused on HPV-related tumors, as well as a better understanding of which patients benefit from this strategy, warrants further investigation.

Study Overview

• Status: Recruiting
• Conditions: Anal Canal Cancer; HPV-Related Carcinoma; HPV-Related Cervical Carcinoma; Uterine Cervical Cancer; HPV-Related Anal Squamous Cell Carcinoma
• Intervention / Treatment: Diagnostic test: ctDNA test; Drug: Pembrolizumab

Detailed Description

The ANA study is a research project aimed at enhancing the treatment and outcomes for patients with cervical and anal canal cancer by using innovative diagnostic and therapeutic methods. The study consists of the following phases:

• Patient identification and selection;
• Recruitment of patients diagnosed with cervical or anal canal cancer who are candidates for treatment with radiotherapy (RT), with or without chemotherapy: patients will be selected based on specific criteria to ensure a representative cohort;
• Development and validation of the ctDNA HPV Test: development of a sensitive and specific test to detect HPV DNA in the blood. This test will undergo rigorous validation to ensure its accuracy and reliability;
• ctDNA monitoring: blood samples collection from patients during treatment and follow-up. ctDNA levels will be monitored in real-time to early detection of residual or recurrent disease. This non-invasive method aims to provide a more accurate assessment of treatment efficacy and disease progression. The results of ctDNA will be compared with traditional imaging methods.
• Complementary immunotherapy treatment: patients with positive ctDNA results after (chemo)radiotherapy will be considered for additional immunotherapy. This phase will evaluate the benefits of combining immunotherapy with standard (chemo)radiotherapy in order to improve patient outcomes;
• Follow-up and outcome evaluation: long-term follow-up of patients to assess clinical outcomes, including survival and quality of life.

The ANA study aims to set new standards in the follow-up and management of HPV-related cervical and anal canal cancer by improving patient care within the Brazilian public health system (SUS).

• Study Type: Interventional
• Enrollment (Estimated): 150
• Phase: Phase 2

Contacts and Locations

• Study Contact: Name: Research Center, Assistant; Phone Number: +55 11 3893-3566; Email: icesp.pesqclinica@hc.fm.usp.br

Study Locations

• Brazil
  • São Paulo, Brazil, 01246-000
    • Recruiting
    • Instituto do Cancer do Estado de Sao Paulo - ICESP
    • Contact: Research Center, Assistant; Phone Number: +55 11 3893-3566; Email: icesp.pesqclinica@hc.fm.usp.br
    • Principal Investigator: Maria DP Estevez Diz, Doctor
    • Sub-Investigator: Camita MV Moniz, Doctor
    • Sub-Investigator: Leticia V Leis, Doctor
    • Sub-Investigator: Pedro Hashizume, Doctor

Participation Criteria

Eligibility Criteria

• Ages Eligible for Study: Adult; Older Adult
• Accepts Healthy Volunteers: No

Description

Inclusion Criteria:

1. Histological diagnosis of anal canal or cervical cancer.
2. Documented presence of HPV.

3. Locally confined or locally advanced disease, defined as:

   1. Anal canal carcinoma stage I to III, according to American Joint Committee on Cancer (AJCC) 8th edition;
   2. Cervical carcinoma stage I B2 to IV A, according to AJCC 8th edition.
4. Indication for definitive treatment with radiotherapy, with or without concomitant chemotherapy.
5. Eastern Cooperative Oncology Group (ECOG)-Performance Status (PS) 0 - 1.
6. Age ≥ 18 years.
7. Signing of the Informed Consent Form (ICF).
8. HIV-positive patients may be included if Cluster of Differentiation 4(CD4) count is greater than or equal to 200.
9. Patients may participate in other concurrent studies, as long as they do not involve interventions related to the treatment of the underlying cancer.

Exclusion Criteria:

1. Patients with unequivocal distant metastasis at diagnosis.
2. For participants with positive ctDNA after treatment, those candidates for participation in Phase II will be excluded if there is unequivocal radiological progression in the first imaging exam after the completion of radiotherapy (with or without chemotherapy) or routine indication for salvage surgery immediately after the conclusion of definitive treatment.
3. Need for recurrent blood transfusions, such as weekly frequency.
4. Another uncontrolled disease representing a life risk, as determined by medical judgment.
5. Personal history of another active invasive malignant neoplasm in the last 5 years, except for non-melanoma skin carcinomas and in situ carcinomas.
6. Pregnant individuals.
7. Active opportunistic infection or disease.
8. History of autoimmune diseases.

Study Plan

How is the study designed?

Design Details

• Primary Purpose: Treatment
• Allocation: Non-Randomized
• Interventional Model: Sequential Assignment
• Masking: None (Open Label)

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Other: Active laboratory monitoring; Phase I Monitoring through collection of laboratory tests	Diagnostic test: ctDNA test; ctDNA involves the collection of peripheral blood samples for the analysis of circulating tumor DNA (ctDNA). The samples are processed using next-generation sequencing (NGS) and/or digital polymerase chain reaction (PCR) techniques to detect specific genetic alterations related to the tumor. The objective is to assess the presence and quantity of ctDNA, providing information on tumor burden and treatment response.
Experimental: Intervention with Immunotherapy; Phase II for participants who are ctDNA positive after standard treatment. Intervention will be with Pembrolizumab	Drug: Pembrolizumab; Participants will receive the institution's standard treatment during Phase I. If ctDNA remains positive between 8 and 12 weeks after the standard treatment, the participant will be invited to proceed to Phase II, which will consist of intravenous immunotherapy for up to 12 months, or until disease progression or unacceptable toxicity occurs. Continuous monitoring with ctDNA testing will be performed during Phase II.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
General Objective	The objective of this study is to develop and validate a low-cost ctDNA test focused on detecting HPV-associated ctDNA for use in patients within the Brazilian Unified Health System (SUS). The test will be evaluated for its effectiveness in improving traditional monitoring methods and guiding the intensification of adjuvant treatment for anogenital neoplasms, including anal canal and cervical cancer. The impact of the test will be measured by correlating ctDNA detection with clinical outcomes of patients over time.	2 years

Other Outcome Measures

Outcome Measure	Measure Description	Time Frame
Number of HPV-focused ctDNA tests developed and validated	Measure the number of HPV-related ctDNA tests developed and validated for detecting HPV ctDNA in patients with anogenital neoplasms.	2 years
Performance of the validated HPV-related ctDNA test	Measure the accuracy and specificity of the validated HPV-related ctDNA test, including the assessment of HPV typing, ctDNA, and HPV E6*I messenger ribonucleic acid (mRNA) levels in the study population.	2 years
Comparison of accuracy between standard ctDNA and HPV-focused ctDNA	valuate the accuracy of standard ctDNA compared to HPV-focused ctDNA and in relation to standard radiological control, based on routine imaging, through serial testing of ctDNA and HPV ctDNA after completion of treatment with definitive radiotherapy or chemoradiotherapy.	2 years
Comparison with Commercially Available HPV ctDNA Tests	Compare the diagnostic accuracy of standard ctDNA with HPV-focused ctDNA and standard radiological control, using routine imaging and serial ctDNA tests after definitive treatment with radiotherapy or chemoradiotherapy.	2 years
Comparison of performance between the locally developed HPV ctDNA test and commercially available tests	Compare the sensitivity, specificity, and overall performance of the locally developed HPV ctDNA test with commercially available tests on the market.	2 years
Direct costs of the ctDNA test	Measurement of the costs associated with performing the ctDNA test, including materials, labor, and technology used.	2 years
Disease progression reduction rate with the use of immunotherapy	Measure the rate of disease progression reduction in patients with positive ctDNA after definitive treatment, in response to the use of immunotherapy.	2 years
Influence of PD-L1 expression and HPV type on immunotherapy response	Relationship between PD-L1 expression levels and HPV types on the immunotherapy response in patients with positive ctDNA. PD-L1 levels will be quantified using immunohistochemistry, and the HPV types present in tumor samples will be categorized. The outcomes will include the treatment response rate and progression-free survival, recorded over a two-year period.	2 years
Correlation between PD-L1 expression and HPV type with immunotherapy response	Measure the correlation between PD-L1 expression and HPV type with the response rate to immunotherapy in patients with positive ctDNA, quantifying the impact of these variables on the effectiveness of immunotherapy.	2 years

Collaborators and Investigators

• Sponsor: Instituto do Cancer do Estado de São Paulo
• Collaborators: Conselho Nacional de Desenvolvimento Científico e Tecnológico
• Investigators: Principal Investigator: Maria DP Estevez Diz, Doctor, Oncologist

Publications and helpful links

General Publications

• Bettegowda C, Sausen M, Leary RJ, Kinde I, Wang Y, Agrawal N, Bartlett BR, Wang H, Luber B, Alani RM, Antonarakis ES, Azad NS, Bardelli A, Brem H, Cameron JL, Lee CC, Fecher LA, Gallia GL, Gibbs P, Le D, Giuntoli RL, Goggins M, Hogarty MD, Holdhoff M, Hong SM, Jiao Y, Juhl HH, Kim JJ, Siravegna G, Laheru DA, Lauricella C, Lim M, Lipson EJ, Marie SK, Netto GJ, Oliner KS, Olivi A, Olsson L, Riggins GJ, Sartore-Bianchi A, Schmidt K, Shih lM, Oba-Shinjo SM, Siena S, Theodorescu D, Tie J, Harkins TT, Veronese S, Wang TL, Weingart JD, Wolfgang CL, Wood LD, Xing D, Hruban RH, Wu J, Allen PJ, Schmidt CM, Choti MA, Velculescu VE, Kinzler KW, Vogelstein B, Papadopoulos N, Diaz LA Jr. Detection of circulating tumor DNA in early- and late-stage human malignancies. Sci Transl Med. 2014 Feb 19;6(224):224ra24. doi: 10.1126/scitranslmed.3007094.
• Jeannot E, Becette V, Campitelli M, Calmejane MA, Lappartient E, Ruff E, Saada S, Holmes A, Bellet D, Sastre-Garau X. Circulating human papillomavirus DNA detected using droplet digital PCR in the serum of patients diagnosed with early stage human papillomavirus-associated invasive carcinoma. J Pathol Clin Res. 2016 Jun 28;2(4):201-209. doi: 10.1002/cjp2.47. eCollection 2016 Oct.
• Bernard-Tessier A, Jeannot E, Guenat D, Debernardi A, Michel M, Proudhon C, Vincent-Salomon A, Bieche I, Pierga JY, Buecher B, Meurisse A, Francois E, Cohen R, Jary M, Vendrely V, Samalin E, El Hajbi F, Baba-Hamed N, Borg C, Bidard FC, Kim S. Clinical Validity of HPV Circulating Tumor DNA in Advanced Anal Carcinoma: An Ancillary Study to the Epitopes-HPV02 Trial. Clin Cancer Res. 2019 Apr 1;25(7):2109-2115. doi: 10.1158/1078-0432.CCR-18-2984. Epub 2018 Nov 30.
• Cabel L, Bonneau C, Bernard-Tessier A, Hequet D, Tran-Perennou C, Bataillon G, Rouzier R, Feron JG, Fourchotte V, Le Brun JF, Benoit C, Rodrigues M, Scher N, Minsat M, Legrier ME, Bieche I, Proudhon C, Sastre-Garau X, Bidard FC, Jeannot E. HPV ctDNA detection of high-risk HPV types during chemoradiotherapy for locally advanced cervical cancer. ESMO Open. 2021 Jun;6(3):100154. doi: 10.1016/j.esmoop.2021.100154. Epub 2021 May 19.
• Jeannot E, Latouche A, Bonneau C, Calmejane MA, Beaufort C, Ruigrok-Ritstier K, Bataillon G, Larbi Cherif L, Dupain C, Lecerf C, Popovic M, de la Rochefordiere A, Lecuru F, Fourchotte V, Jordanova ES, von der Leyen H, Tran-Perennou C, Legrier ME, Dureau S, Raizonville L, Bello Roufai D, Le Tourneau C, Bieche I, Rouzier R, Berns EMJJ, Kamal M, Scholl S. Circulating HPV DNA as a Marker for Early Detection of Relapse in Patients with Cervical Cancer. Clin Cancer Res. 2021 Nov 1;27(21):5869-5877. doi: 10.1158/1078-0432.CCR-21-0625. Epub 2021 Jul 1.
• Lefevre AC, Pallisgaard N, Kronborg C, Wind KL, Krag SRP, Spindler KG. The Clinical Value of Measuring Circulating HPV DNA during Chemo-Radiotherapy in Squamous Cell Carcinoma of the Anus. Cancers (Basel). 2021 May 18;13(10):2451. doi: 10.3390/cancers13102451.

Study record dates

Study Major Dates

• Study Start (Actual): March 14, 2025
• Primary Completion (Estimated): December 1, 2026
• Study Completion (Estimated): January 1, 2027

Study Registration Dates

• First Submitted: October 4, 2024
• First Submitted That Met QC Criteria: October 10, 2024
• First Posted (Actual): October 15, 2024

Study Record Updates

• Last Update Posted (Actual): December 24, 2025
• Last Update Submitted That Met QC Criteria: December 18, 2025
• Last Verified: December 1, 2025

More Information

Terms related to this study

• Keywords: Biomarkers; Diagnostic test; ctDNA; HPV; Molecular diagnosis; Cervical tumors; Anal canal tumors
• Additional Relevant MeSH Terms: Urogenital Diseases; Genital Diseases; Urogenital Neoplasms; Neoplasms by Site; Neoplasms; Female Urogenital Diseases; Female Urogenital Diseases and Pregnancy Complications; Uterine Diseases; Genital Diseases, Female; Genital Neoplasms, Female; Uterine Cervical Diseases; Uterine Neoplasms; Uterine Cervical Neoplasms; Anal Canal Carcinoma; Molecular Mechanisms of Pharmacological Action; Antineoplastic Agents; Antineoplastic Agents, Immunological; Immune Checkpoint Inhibitors; pembrolizumab
• Other Study ID Numbers: NP5125/2024; 79795024.8.0000.0068 (Registry Identifier: Certificate of Ethical Review Submission); 444027/2023-8 (Other Grant/Funding Number: CNPq)

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: NO

IPD Plan Description

It has been decided not to share individual participant data (IPD) related to the study for several reasons. Protecting the privacy of participants is a priority. Sharing IPD may expose sensitive information that, even when de-identified, can be traced back to individuals.

Furthermore, the complexity of the data makes sharing challenging without the risk of misunderstandings or misinterpretations, which could compromise the integrity of the research. Sharing IPD without the explicit consent of participants may violate ethical principles of respect and protection.

There is also a need to comply with regulatory guidelines governing data sharing to avoid potential legal issues. Finally, the focus will be on disseminating aggregated results that can benefit the scientific community and the public without compromising individual privacy.

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No
• product manufactured in and exported from the U.S.: No