Determining Minimal Residual Disease Using ctDNA Deep Sequencing (CIT-SEQ)

Determination of Minimal Residual Disease by Deep ctDNA Sequencing.

Preoperative neoadjuvant chemotherapy is the standard treatment for locally advanced gastrointestinal tumours. However, not all patients respond to preoperative treatment. Early identification of progression during neoadjuvant chemotherapy or diagnosis of early disease relapse during adjuvant treatment is essential to modify the treatment strategy. The aim of this project is to validate ctDNA as a biomarker of molecular relapse/progression of disease.

Study Overview

• Status: Recruiting
• Conditions: Gastric Cancer; Pancreatic Cancer; Esophageal Adenocarcinoma
• Intervention / Treatment: Diagnostic test: NGS sequencing of mutations selected based on sequencing of primary tumors

Detailed Description

Neoadjuvant chemotherapy is the standard of care for locally advanced gastrointestinal tumors. However, not all patients respond to preoperative treatment. Early identification of disease progression during neoadjuvant chemotherapy or diagnosis of early disease relapse during adjuvant therapy is essential for modifying the treatment strategy. The aim of the project is to validate ctDNA as a molecular biomarker of disease relapse/progression.

The persistence of tumor cells after primary treatment of cancer is a key prognostic indicator of the disease's future course. In solid tumors, minimal residual disease (MRD) may involve tumor cells in the blood, small metastases in the body, or a small portion of the primary tumor that remains after treatment. Detection of MRD at a stage when the tumor cannot be detected by morphological imaging techniques is becoming an important diagnostic indicator that could better stratify patients after primary treatment in the future. MRD determination should thus serve in the future both to indicate further treatment and to monitor treatment response. For the successful clinical application of MRD determination, it is crucial to establish a sufficiently sensitive and specific method based on the detection of circulating tumor DNA (ctDNA). Tumor ctDNA is a component of circulating free DNA (cfDNA), which is released into the plasma from apoptotic or necrotic cells in the form of fragments with an average length of approximately 170 bp. The release of DNA from cells into the bloodstream can be influenced by a number of factors, which is reflected in the high variability of total cfDNA concentration, which can range from 1 to 100,000 fragments per milliliter of plasma. Therefore, to quantify MRD, it is crucial to determine both the total concentration of cfDNA and the proportion of tumor ctDNA. To reliably distinguish tumor ctDNA from non-tumor cfDNA, the detection of mutations identified in the primary tumor is currently used. For this purpose, PCR or digital PCR detecting specific mutations using fluorescent probes can be used. An alternative to PCR is mutation detection using next-generation sequencing (NGS), which allows for the detection of multiple different mutations in parallel. A disadvantage of MRD determination using commercial NGS kits designed for liquid biopsies is the high financial cost and the time-consuming preparation of sequencing libraries. We hypothesize that deep sequencing of cfDNA targeting mutations identified in the primary tumor can significantly increase both the specificity and sensitivity of MRD detection from liquid biopsies. Deep sequencing of 2-3 genomic regions with known mutations should enable the parallel detection of MRD in multiple patients while reducing the cost of this type of testing compared to commercially available options.

• Study Type: Interventional
• Enrollment (Estimated): 34
• Phase: Not Applicable

Contacts and Locations

• Study Contact: Name: Martina Lojová, PhD; Phone Number: +420543136232; Email: martina.lojova@mou.cz
• Study Contact Backup: Name: Tereza Štěpánková, PhD; Phone Number: +420543136223; Email: tereza.stepankova@mou.cz

Study Locations

• Czechia
  • Brno, Czechia, 65653
    • Recruiting
    • Masaryk Memorial Cancer Institute

Participation Criteria

Eligibility Criteria

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

Description

Inclusion Criteria:

• Consent to participate in the study
• Patients with esophageal, gastric, or pancreatic cancer, stage 0-2
• Patients with locally advanced, potentially operable disease treated with systemic perioperative chemotherapy or chemoradiotherapy
• Patients with metastatic disease treated with first- to third-line palliative systemic therapy

Exclusion Criteria:

• not specified

Study Plan

How is the study designed?

Design Details

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

Number of Arms

2

Arms and Interventions

Participant Group / Arm	Intervention / Treatment
Experimental: Interventional; Participants with locally advanced potentially resectable esophageal, gastric, or pancreatic cancer receiving neoadjuvant/perioperative treatment will undergo serial blood sampling for plasma isolation and circulating tumor DNA (ctDNA) analysis. Samples will be collected prior to initiation of neoadjuvant therapy, at the time of surgery, and subsequently at approximately 3-month intervals for 1-2 years after surgery or until disease progression. ctDNA analyses will be performed using a targeted next-generation sequencing (NGS) approach to evaluate longitudinal changes in tumor-specific genomic alterations and their association with treatment response and disease recurrence.	Diagnostic test: NGS sequencing of mutations selected based on sequencing of primary tumors; Sequencing of the primary tumor will be performed on a MiSeq instrument using the Accel-Amplicon panel, which covers the most common mutations. For deep sequencing of ctDNA, 2-3 genomic regions carrying mutations in the primary tumor will be selected for the patient. For these regions, sets of universal primers will be designed to amplify the mutations most commonly found in solid tumors (RAS, BRAF, TP53, EGFR, APC, etc.). Another set of universal primers will contain a sequence for indexing individual samples, enabling the parallel sequencing of up to 96 samples simultaneously. The sequencing library will be analyzed on a NextSeq 500 instrument, which allows for up to 400 million reads in a single sequencing run. The sequencing results will then be correlated with the clinical course of the disease.
Active Comparator: Control; Participants with esophageal, gastric, or pancreatic cancer receiving first-line palliative systemic therapy will undergo serial blood sampling for plasma isolation and ctDNA analysis. Samples will be collected prior to initiation of systemic treatment and subsequently at approximately 3-month intervals during first-line therapy. ctDNA analyses will be performed using the same targeted NGS methodology to assess longitudinal changes in tumor-specific genomic alterations during systemic treatment.	Diagnostic test: NGS sequencing of mutations selected based on sequencing of primary tumors; Sequencing of the primary tumor will be performed on a MiSeq instrument using the Accel-Amplicon panel, which covers the most common mutations. For deep sequencing of ctDNA, 2-3 genomic regions carrying mutations in the primary tumor will be selected for the patient. For these regions, sets of universal primers will be designed to amplify the mutations most commonly found in solid tumors (RAS, BRAF, TP53, EGFR, APC, etc.). Another set of universal primers will contain a sequence for indexing individual samples, enabling the parallel sequencing of up to 96 samples simultaneously. The sequencing library will be analyzed on a NextSeq 500 instrument, which allows for up to 400 million reads in a single sequencing run. The sequencing results will then be correlated with the clinical course of the disease.

What is the study measuring?

Primary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Sensitivity of targeted NGS assay for ctDNA mutation detection	Proportion of tumor-confirmed driver mutations detected in plasma cfDNA using the developed targeted NGS assay. Unit of Measure: Percentage (%)	Through study completion, an average of 24 months.

Secondary Outcome Measures

Outcome Measure	Measure Description	Time Frame
Monitoring ctDNA levels	A secondary objective is to monitor ctDNA levels throughout the patient's treatment and to determine appropriate intervals for plasma collection after surgery and during adjuvant therapy.	Through study completion, an average of 24 months.
Variant allele frequency (VAF) of detected driver mutations in cfDNA	Quantitative assessment of tumor-specific mutations detected in cfDNA using targeted NGS sequencing. Unit of Measure: Variant allele frequency (%)	Through study completion, an average of 24 months.

Collaborators and Investigators

• Sponsor: Masaryk Memorial Cancer Institute
• Investigators: Study Director: Petr Müller, MD, PhD, Masaryk Memorial Cancer Institute; Principal Investigator: Radka Lordick Obermannová, MD, Doc, PhD, Masaryk Memorial Cancer Institute

Study record dates

Study Major Dates

• Study Start (Actual): August 22, 2022
• Primary Completion (Estimated): December 31, 2026
• Study Completion (Estimated): December 31, 2026

Study Registration Dates

• First Submitted: June 10, 2026
• First Submitted That Met QC Criteria: June 16, 2026
• First Posted (Actual): June 22, 2026

Study Record Updates

• Last Update Posted (Actual): June 24, 2026
• Last Update Submitted That Met QC Criteria: June 19, 2026
• Last Verified: June 1, 2026

More Information

Terms related to this study

• Keywords: NGS sequencing; Minimal residual disease; gastrointestinal tumors; Preoperative neoadjuvant chemotherapy
• Additional Relevant MeSH Terms: Endocrine System Diseases; Pathologic Processes; Neoplasms by Site; Neoplasms; Gastrointestinal Neoplasms; Digestive System Diseases; Gastrointestinal Diseases; Stomach Diseases; Endocrine Gland Neoplasms; Pancreatic Diseases; Neoplastic Processes; Pathological Conditions, Signs and Symptoms; Stomach Neoplasms; Neoplasm, Residual; Pancreatic Neoplasms; Digestive System Neoplasms; Adenocarcinoma Of Esophagus
• Other Study ID Numbers: A2/23

Plan for Individual participant data (IPD)

• Plan to Share Individual Participant Data (IPD)?: YES
• IPD Plan Description: IPD to be shared in pseudonymized form during the study. IPD to be published in anonymized form.
• IPD Sharing Time Frame: after study completion

IPD Sharing Access Criteria

During the study, data will be managed in pseudonymized form in a protected database environment, available only for study team.

After completion of the study, the data will be fully anonymized for publication purposes. All publication outputs of the study will be carried out by a team of researchers led by the principal investigator. The submission of each publication is subject to the approval of the principal investigator.

The results of this study may be published or presented at scientific meetings after approval by the PI and always after anonymization of the subjects' personal data in accordance with Act No. 101/2000 Coll., on the protection of personal data.

• IPD Sharing Supporting Information Type: CSR

Drug and device information, study documents

• Studies a U.S. FDA-regulated drug product: No
• Studies a U.S. FDA-regulated device product: No