Clinical trial NCT05680077: KCNA3 and OTOP2 Gene Methylation Combined Detection Kit (Fluorescent PCR Method)
Official title: Methylation Detection of KCNA3 and OTOP2 Genes in Plasma for the Auxiliary Diagnosis of Esophageal Cancer/Precancerous Lesions: a Clinical Trial
Study type: OBSERVATIONAL
Overall status: COMPLETED
Conditions: Esophageal Neoplasms
Keywords: Esophageal carcinoma
Esophageal high-grade intraepithelial neoplasia
methylation
Fluorescent PCR
Lead sponsor: Wuhan Ammunition Life-tech Co., Ltd
Sponsor class: INDUSTRY
Collaborators: name: Changhai Hospital
First posted: 2023-01-11
Enrollment: 1116
Sex: ALL
Minimum age (years): 40
Standard ages: ADULT
OLDER_ADULT
Accepts healthy volunteers: no
Observational model: CASE_CONTROL
Time perspective: CROSS_SECTIONAL
Sampling method: PROBABILITY_SAMPLE
Study population: 1. Esophageal cancer patients, number ≥ 300, containing different TNM stages.
2. Esophageal high-grade intraepithelial neoplasis patients, number ≥70.
3. Patients with benign diseases of digestive system including esophagitis, gastritis, enteritis, appendicitis, gastric polyps, colorectal polyps, etc. , number ≥200.
4. Patients with other digestive system malignancies (including gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer, etc.) and patients with non-digestive system malignancies (including thyroid cancer, lung squamous cell carcinoma, cervical cancer, endometrial cancer, breast cancer, prostate cancer, etc.)， number ≥350.
5. All the enrolled participants should be ≥1000.

Brief summary: The goal of this observational study is to test the clinical efficacy of "KCNA3 and OTOP2 gene methylation combined detection kit (fluorescence PCR method)"in esophageal cancer and high-grade esophageal neoplasia auxiliary diagnosis.The main questions it aims to answer are:

1. How consistent are the test results of the kit with the clinical reference diagnostic criteria?
2. Sanger sequencing can visually show whether each sample contains methylation sites, so in this clinical trial, the kit results were compared with Sanger sequencing results to analyze the reagent's accuracy in detecting KCNA3 and OTOP2 gene methylation.

Each participant is required to provide no less than 10 ml of blood to complete the kit test.

Detailed description: Esophageal carcinoma is caused by abnormal hyperplasia of the squamous or glandular epithelium, the main pathological types are squamous cell carcinoma and adenocarcinoma. Globally, esophageal cancer has the seventh highest incidence and the sixth leading cause of cancer death. Statistics showed that in 2020, there were 604,000 new cases of esophageal cancer and 54.4 deaths.

The 5-year survival rates were significantly different in esophageal carcinomas of different stages, for example, it's 84.9% for stage Ia, 70.9% for stage Ib, 56.2% for stage IIa, 43.3% for stage IIb, 37.9% for stage IIIa, 23.3% for stage IIIb, 12.9% for stage IIIc and 3.4% for stage IV. Therefore, early diagnosis and treatment is most effective strategy to reduce the incidence and mortality of esophageal cancer.

Endoscopy and pathological biopsy are currently the "golden standard" for diagnosing esophageal cancer, however, limited by medical resources, tolerance and other issues, population screening by endoscopy is not cost-effective and applicable. There is no recognized and effective serologic marker for the detection of early esophageal carcinoma and precancerous lesions, the effect of multiple serological markers combined detection is better than that of single markers, and the accuracy were 74%\~85% , but it is still suboptimal.

Methylation of the tumor suppressor gene promoter region CpG island is the most common change during tumorgenesis, studies showed that methylation events could occur at the early stages of esophageal cancer.

Circulating tumor DNA (ctDNA) is derived from DNA fragments produced by apoptosis, necrosis or secretion of tumor cells, which is a part of circulating free (cfDNA). CtDNA contains the same gene defects as its source tumor DNA, such as point mutation, rearrangement, amplification, microsatellite change, epigenetic modification, etc.

KCNA3 is a member of the protein family that encodes potassium channels and mainly affects cell viscosity. OTOP2 is a member of the protein family encoding proton transport channels, some studies have shown that the methylation levels of two genes in esophageal cancer tissues were significantly higher than that in adjacent tissues, and they can be used as a potential marker for the diagnosis of esophageal cancer.

This kit is based on real-time fluorescence PCR technology to achieve the qualitative detection of KCNA3 and OTOP2 methylation in plasma samples. Before PCR amplification, cfDNA was transformed by bisulfite and ACTB (β-Actin) gene is used us the internal reference.

Eligibility criteria: Inclusion Criteria:

1. esophageal carcinoma patients confirmed or highly suspected by endoscopy, imaging examination or pathological biopsy, and high-grade intraepithelial neoplasia of the esophagus patients mainly enrolled from individuals who planned to undergo radical esophagectomy of esophageal cancer, endoscopic submucosal dissection or primary chemoradiotherapy.
2. patients with benign diseases of digestive system who have undergone endoscopy (including esophagitis, gastritis, enteritis, appendicitis, gastric polyps, colorectal polyps, etc.).
3. Untreated patients with other digestive system malignancies (including gastric cancer, colorectal cancer, liver cancer, pancreatic cancer, bile duct cancer, etc.) and patients with non-digestive system malignancies (including thyroid cancer, lung squamous cell carcinoma, cervical cancer, endometrial cancer, breast cancer, prostate cancer, etc.) confirmed by clinical reference diagnostic criteria.

Exclusion Criteria:

1. patients who have received anti-tumor therapy such as radiotherapy/chemotherapy;
2. Esophageal carcinoma and high-grade intraepithelial neoplasia patients who were also suffering from other malignant tumors;
3. Samples not kept as required or samples of hemolysis;
4. The sample volume fails to meet the detection requirements;
5. Esophageal carcinoma patients with distant metastasis.

Primary outcomes:
measure: validity
timeFrame: Immediately after the procedure
Secondary outcomes:
measure: reliability
timeFrame: Immediately after the procedure
Other outcomes:
Locations:
city: Shanghai
country: China
facility: The First Affiliated Hospital of Naval Military Medical University
Study officials:
affiliation: Changhai Hospital
name: Luowei Wang, MD
role: PRINCIPAL_INVESTIGATOR
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