title: An international collaboration to harmonize the quantitative plasma Epstein-Barr virus DNA assay for future biomarker-guided trials in nasopharyngeal carcinoma.
abstract: OBJECTIVE
  Persistently elevated posttreatment plasma EBV DNA is a robust predictor of relapse in nasopharyngeal carcinoma (NPC). However, assay standardization is necessary for use in biomarker-driven trials. We conducted a study to harmonize the method between four centers with expertise in EBV DNA quantitation.
  
  
  METHODS
  Plasma samples of 40 patients with NPC were distributed to four centers. DNA was extracted and EBV DNA copy number was determined by real-time quantitative PCR (BamHI-W primer/probe). Centers used the same protocol but generated their own calibrators. A harmonization study was then conducted using the same calibrators and PCR master mix and validated with ten pooled samples.
  
  
  RESULTS
  The initial intraclass correlations (ICC) for the first 40 samples between each center and the index center were 0.62 [95% confidence interval (CI): 0.39-0.78], 0.70 (0.50-0.83), and 0.59 (0.35-0.76). The largest variability was the use of different PCR master mixes and calibrators. Standardization improved ICC to 0.83 (0.5-0.95), 0.95 (0.83-0.99) and 0.96 (0.86-0.99), respectively, for ten archival frozen samples. For fresh plasma with spiked-in EBV DNA, correlations were more than 0.99 between the centers. At 5 EBV DNA copies per reaction or above, the coefficient of variance (CV) was less than 10% for the cycle threshold (Ct) among all centers, suggesting this concentration can be reliably used as a cutoff for defining the presence of detectable EBV DNA.
  
  
  CONCLUSIONS
  Quantitative PCR assays, even when conducted in experienced clinical labs, can yield large variability in plasma EBV DNA copy numbers without harmonization. The use of common calibrators and PCR master mix can help to reduce variability.
authors: Department of Radiation Oncology, Stanford University, 875 Blake Wilbur Dr., MC 5847, Stanford, CA 94305, USA. Qle@stanford.edu
authors: Quynh-Thu
authors: QT
authors: Le
authors: Qiang
authors: Q
authors: Zhang
authors: Hongbin
authors: H
authors: Cao
authors: Ann-Joy
authors: AJ
authors: Cheng
authors: Benjamin A
authors: BA
authors: Pinsky
authors: Ruey-Long
authors: RL
authors: Hong
authors: Joseph T
authors: JT
authors: Chang
authors: Chun-Wei
authors: CW
authors: Wang
authors: Kuo-Chien
authors: KC
authors: Tsao
authors: Ym Dennis
authors: YD
authors: Lo
authors: Nancy
authors: N
authors: Lee
authors: K Kian
authors: KK
authors: Ang
authors: Anthony T C
authors: AT
authors: Chan
authors: K C Allen
authors: KC
authors: Chan
mesh_terms: D014408:Biomarkers, Tumor / Q000097:blood / Q000235:genetics / Q000592:standards; D002138:Calibration / Q000592:standards; D002277:Carcinoma; D004279:DNA, Viral / Q000097:blood* / Q000235:genetics / Q000592:standards; D020031:Epstein-Barr Virus Infections / Q000097:blood / Q000175:diagnosis / Q000821:virology*; D004854:Herpesvirus 4, Human / Q000235:genetics*; D006801:Humans; D007391:International Cooperation; D007753:Laboratories / Q000592:standards; D000077274:Nasopharyngeal Carcinoma; D009303:Nasopharyngeal Neoplasms / Q000097:blood / Q000821:virology*; D060888:Real-Time Polymerase Chain Reaction / Q000379:methods / Q000592:standards; D012015:Reference Standards; D015203:Reproducibility of Results; D012680:Sensitivity and Specificity
chemical_list: D014408:Biomarkers, Tumor; D004279:DNA, Viral