Assessing Systemic Treatment and ctDNA Monitoring Efficacy in Metastatic Cancer Patients Using K4Care Testing.

Evaluation of Systemic Therapy and the Effectiveness of Circulating Tumor DNA (ctDNA) Assays in Monitoring Treatment Response in Patients With Metastatic Cancer Undergoing K4Care Testing

The goal of this observational study is to investigate the real-world treatment landscape of patients with metastatic cancer and determine the role of ctDNA in monitoring treatment response in patients with metastatic cancer who have undergone K4Care testing

Study design: This is a prospective, longitudinal, non-interventional study. Samples will be collected from patients with metastatic cancer who have undergone K4Care testing at Gene Solutions from 03/2025 to 03/2026.

- Definition:

• Progression-free survival (PFS): The time from the start of treatment until disease progression, defined as the appearance of new tumor lesions or the spread of existing lesions to other organs or body parts.
• Overall survival (OS): The time from the start of the study (for non-randomized clinical trials) until death from any cause.
• Cell-free DNA (cfDNA) refers to DNA fragments released from cells into the bloodstream, with an average size of approximately 170 bp. A recent study showed that the concentration of cfDNA in the serum of cancer patients is elevated compared to healthy individuals. Circulating tumor DNA (ctDNA) refers to cfDNA released from cancer cells and carries tumor-associated mutations. The proportion of ctDNA within total cfDNA in cancer patients is highly variable, ranging from over 25% to as low as 0.01% (meaning that for every 10,000 cfDNA molecules in the blood, only 1 ctDNA molecule is released from cancer cells). This ratio is also known as the mutation allele fraction (MAF). MAF varies depending on the type and stage of cancer. The later the stage of the disease, the larger the tumor, and the more ctDNA is released into the blood. The presence of ctDNA in the serum indicates the presence of cancer in the body at the cellular level.

Due to the specificity of ctDNA for cancer, the correlation between ctDNA concentration and tumor size is less affected by other factors. Numerous studies have demonstrated that ctDNA levels change during treatment and correlate with early tumor response, which can be used as a companion biomarker and complement imaging response monitoring in patients treated with immunotherapy. Therefore, many studies have used ctDNA as a potential marker in monitoring tumor response in radical surgery, neoadjuvant chemoradiation, and evaluating treatment response in metastatic patients. NGS technology has brought tremendous value to the identification and treatment of cancer in recent years, helping to prolong the survival of patients and support the development of appropriate treatments for each patient with specific mutations. In addition, gene sequencing technology allows for the accurate detection of cancer-causing mutations in cfDNA samples, thereby identifying the composition of extracellular ctDNA released from the tumor.

• Blood sample: Each participant will have two times of 10mL blood sample collections, 1st blood sample collection is on the enrollment date and the 2nd blood sample collection is on the follow up visit date, from 3-6 months after the enrollment date or according to any participant follow-up visit during the study conduct.

• Clinical information to be collected: will be collected and supplemented (if missing) during the patient's follow-up visits as scheduled by the physician. The information to be collected includes:

  1. Basic patient demographics and medical history: age; sex; medical history (personal/family, viral infection status); diagnosis: time of diagnosis, cancer type (primary, secondary, metastatic), stage, location, histology, and other biochemical tests.

  2. Pathological results, immunohistochemical results, and biopsy site.

  3. Treatment regimen information: treatment types (surgery/chemotherapy/radiotherapy/targeted therapy), time points (start, mid-treatment, end of treatment):

     • Curative surgery
     • TKI/ICI or Chemotherapy for metastatic stage
     • Palliative treatment for metastatic stage

  4. Patient status: RECIST 1.1 response assessment at time points of Progressive Disease, Stable Disease, Partial Response, and Complete Response, based on the clinical physician's assessment documented in the medical record and accompanied by imaging (if available).

     • Time of progression and metastatic site
     • Survival status (alive or deceased). Cause of death. Time of death.
     • Information regarding death: the cause of death to evaluate whether it is related to cancer complications.

  5. Time of CT/MRI imaging, results, and clinical physician's interpretation (images can be provided if available)

Inclusion Criteria:

• Pathological and imaging confirmation of metastatic or progressive stage IV cancer.
• Consent to the use of their clinical data.
• Diagnosis of one of the following common solid tumor types: lung, colorectal, breast, gastric, or cancer of unknown primary origin.
• Prior K4Care testing to identify tumor tissue (FFPE) mutations and consent to the use of their sequencing data.

Exclusion Criteria:

• Refusal to participate in the study.
• Absence of prior K4Care testing.
• Diagnosis of early-stage cancer or hematologic malignancy.
• Refusal to provide clinical information or blood samples.