Paxgene blood ccfdna tube

10 mL blood samples were collected from patients from a peripheral vein or central line in PAXgene blood ccfDNA tubes (768165, BD Biosciences). Samples were centrifuged at 2000g for 15 min to isolate plasma, followed by a second spin at 2000g for 10 min to remove any remaining cells. Buffy coat was isolated after the first round of centrifugation.
2 mL saliva samples were collected in Oragene self‐collection tubes (OG‐600, DNA genotek) by patients according to the MD Anderson protocol, as previously described by Wang et al.¹⁹ In brief, patients were asked to allow saliva to collect in the floor of the mouth for 5 min before spitting into a collection vial. Upon filling the vial, the self‐collection tubes were closed to release the DNA preservation solution, and were inverted 12 times. Samples were centrifuged at 2000g for 15 min to remove cellular matter from cell‐free saliva. Samples were then centrifuged at 2000g for 10 min. If cellular matter was present after the second centrifugation step, this step was repeated. Samples were stored at −80°C until DNA extraction.

All patients in this study provided written informed consent for the IRB‐approved study (Protocol Number: 700–04 and 900–00) at Kyushu University (Fukuoka, Japan) and its related facilities between January 2018 and April 2020. Twenty‐six patients with histologically confirmed HNSCC were enrolled. Tumors were staged according to the 8th edition of the Union for International Cancer Control TNM classification system, except for external auditory canal (EAC) cancer. EAC cancers were staged according to the modified Pittsburgh classification.¹⁴ Tumor tissues were collected by biopsy before initial treatment or during initial surgery, immediately soaked in Allprotect Tissue Reagent (Qiagen), and then stored at −80°C. Blood samples for cell‐free DNA (cfDNA) were collected using PAXgene Blood ccfDNA Tube (BD Biosciences) and were centrifuged at 1700g for 10 min at room temperature within 3 h after blood collection. Plasma (supernatant) was stored at −80°C until the extraction of cfDNA. Blood samples for PBMCs were collected using BD Vacutainer CPT Cell Preparation Tube (BD Biosciences) and were centrifuged at 1700g for 20 min at room temperature within 3 h after blood collection. PBMCs were collected from the buffy coat layer and stored at −80°C until DNA extraction.