Next-generation Sequencing in Gastrointestinal Cancer
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|ClinicalTrials.gov Identifier: NCT03476057|
Recruitment Status : Not yet recruiting
First Posted : March 23, 2018
Last Update Posted : July 2, 2018
|Condition or disease||Intervention/treatment|
|Gastrointestinal Cancer||Diagnostic Test: Device： NGS sequencing cfDNA|
Sample DNA handling： Peripheral blood lymphocytes (PBLs), and plasma were collected for analysis for each patient. 10mL tubes containing blood samples with EDTA added were centrifuged at 1000g for 10min. The cell pellets containing peripheral blood lymphocytes were stored at -20 °C. The supernatants were centrifuged again at 10,000 g for 10 min, and plasma was collected and stored at -80°C.Tiangen whole blood DNA Kit (Tiangen, Beijing, PRC) were used to extracted DNA from peripheral blood lymphocytes, respectively. QIAamp Circulating Nucleic Acid Kit (Qiagen, German) was used to extract cfDNA form plasma. All kits were used according to the manufacturers' instructions.
Library preparation and sequencing： For each sample, DNA was quantified with the Qubit dsDNA HS Assay kit (Life Technologies,USA) as manufacturer's recommended protocol. Targeted amplification and Illumina adapter-ligated library preparation was performed using Amplicon Sequencing-Illumina Compatible Kit following manufacturer's instructions (Questgenomics, Nanjing, PRC). All samples were subjected to Illumina HiSeq X-Ten for paired-end sequencing (150bp each end). The AmpliSeq Cancer Panel covers 1406 cancer-associated genes which developed by Co. Roche.
Variant calling： Initial data from HiSeq X-Ten were evaluated by using fastQC (v0.11.3). Raw reads were mapped to reference genome hg19 by using BWA (0.7.12-r1039). Program Samtools and VarScan (v2.4.1) was used for variant calling: (1) the average total coverage depth was defined as >1000 and each variant coverage as >10; for called variant, at least one sample with variant frequency >1%, variant frequency of each sample >0.5%, and P value <0.01; (2) visual examination of the mutations was performed using Samtools software (http://samtools.sourceforge.net) and possible errors specific to one DNA strand were filtered out. Software ANNOVAR (v2015-06-17) and snpEff (v4.2) was used for variant annotation
Statistical analysis： For variant frequency less than 0.5%, 0 was replaced. R (hclust, v3.2.4) was used for variant frequency clustering analysis to show what types of samples from cancer patients are more similar. Student's T test was applied for comparison of cfDNA concentration and p<0.05 was considered statistically significant.
|Study Type :||Observational|
|Estimated Enrollment :||200 participants|
|Official Title:||Next-generation Sequencing in Gastrointestinal Cancer|
|Estimated Study Start Date :||July 1, 2018|
|Estimated Primary Completion Date :||December 31, 2020|
|Estimated Study Completion Date :||December 31, 2020|
Advanced gastrointestinal tumor
200 patients with pathologically confirmed Advanced gastrointestinal tumor who never treated with chemotherapy at Fujian Cancer Hospital
Diagnostic Test: Device： NGS sequencing cfDNA
NGS sequencing cfDNA and Tumor Burden
- cfDNA [ Time Frame: 6 months ]demonstrates the spectrum of cfDNA alterations/ profiling
- Tumor mutation burden [ Time Frame: 6 months ]demonstrates the tumor mutation burden alterations/ profiling
Biospecimen Retention: Samples With DNA
To learn more about this study, you or your doctor may contact the study research staff using the contact information provided by the sponsor.
Please refer to this study by its ClinicalTrials.gov identifier (NCT number): NCT03476057
|Contact: Lin Rongbo, Doctorfirstname.lastname@example.org|
|Fuzhou, Fujian, China, 350014|
|Contact: Rongbo Lin, MD 86+13705919382 email@example.com|