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.

Inclusion Criteria:

• 1) histologically confirmed high-grade osteosarcoma;
• 2) older than 10 yrs;
• 3) initially treated in Musculoskeletal Tumor Center of Peking University People's Hospital;
• 4) Serum samples are available;
• 5) completed neo-adjuvant chemotherapy and at least 8 cycles of adjuvant chemotherapy;
• 6) expected to live longer than 3 months with Eastern Cooperative Oncology Group performance status of 0 or 1;
• 7) acceptable hematologic, hepatic, and renal function.

Exclusion Criteria:

• 1) Serum samples are not qualified;
• 2) Patients who could not complete neo-adjuvant chemotherapy or at least 4-month adjuvant chemotherapy;
• 3) lost to follow-up.

Biopsy proved high-grade osteosarcoma, who should be older than 10 years and could complete routine chemo-protocol for osteosarcoma, could get enrolled.

The investigators of this study would like to share IPD. However other cooperative institution's consent will be needed for sharing IPD.