Histologic Response (Tumor Necrosis Rate) Clinical Trial
Official title:
Indentifying Epigenetic Biomarkers From Peripheral Blood of Osteosarcoma Patients Based on hMe-Seal Technique
hMe-Seal is a low-input whole-genome cell-free 5hmC sequencing method based on selective chemical labeling. It uses β-glucosyltransferase (βGT) to selectively label 5hmC with a biotin via an azide-modified glucose for pull-down of 5hmC-containing DNA fragments for sequencing. After selectively constructing 5hmC library, highthroughput-sequencing will be performed on an Illumina Nextseq-500 instrument. By ways of Rawdata processing, differential loci between Osteosarcoma group and control group will be detected to indentify specific epigenetic biomarkers of Osteosarcoma.
The investigator want to enroll 100 osteosarcoma participants initially treated in
Musculoskeletal Tumor Center of Peking University People's Hospital(PKUPH). All those
participants will follow the chemo-protocol for osteosarcoma in PKUPH.8 ml of peripheral
blood would be drawed before each cycle of chemotherapy for further analysis. After
definitive surgery, participants will need to take 8ml of peripheral blood every 2 months for
6 months. A total of 6 times of blood drawing will need to be done.
In hMe-Seal approach, peripheral blood is collected into EDTA-coated Vacutainers. Plasma is
collected from the blood samples after centrifugation at 1 600× g for 10 min at 4 °C and 16
000× g at 10 min at 4 °C. CfDNA is extracted using the Circulating Nucleic Acid Kit(QIAGEN).
After that, cfDNA (1-10 ng) is end repaired, 3'-adenylated and ligated to DNA Barcodes using
KAPA Hyper Prep Kit (Kapa Biosystems). Ligated DNA is incubated in a solution containing
HEPES buffer, UDP-6-N3-Glc and βGT . After that, DBCO-PEG4-biotin is directly added to the
reaction mixture. Next, DNA is purified by Micro Bio-Spin 30 Column (Bio-Rad). The purified
DNA is incubated with M270 Streptavidin beads (Life Technologies) in specific buffer. The
beads are subsequently undergone three 5-min washes each with four kinds of different
buffers. All binding and washing are done at room temperature with gentle rotation. Beads are
then resuspended in water and amplified with 16 cycles of PCR amplification. The PCR products
are purified using AMPure XP beads. Pair-end 38bp sequencing is performed on the NextSeq-500
instrument.
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