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Journal of Clinical Oncology, Vol 23, No 13 (May 1), 2005: pp. 3163-3164 © 2005 American Society of Clinical Oncology. DOI: 10.1200/JCO.2005.05.430
Reproducibility of a Semiquantitative Measurement of Circulating DNA in Plasma From Neoplastic PatientsIstituto Nazionale per lo Studio e la Cura dei Tumori, Milan, Italy To the Editor: The recent editorial by Dr Paul A. Bunn1 commenting on the article by Sozzi et al2 published in the November 1, 2003 issue of the Journal of Clinical Oncology, as well as the many other papers evaluating serum plasma markers in lung cancer patients and controls clearly pinpoints the steps necessary to develop a diagnostic test of clinical use. Plasma collection and storage, DNA isolation, and quantification represent the most critical methodologic and technical aspects related to cell-free DNA determination. Some of these preanalytic and analytic aspects have already been or are presently under investigation.3-4 Several methods, all based on a semiquantitative evaluation, have been used for DNA quantification,5 but none of them has been evaluated in terms of reproducibility. However, in the developing process for a novel biomarker of potential clinical utility, it is fundamental to define assay performance and criteria for interpretation and reporting of results, because lack of standardized and reproducible laboratory methods represents one of the major drawbacks of translating findings from laboratories into clinical practice.1 We proposed to assess the pattern of measurement concordance of an approach currently used to quantify DNA in plasma6 in a series of 15 consecutive patients with intestinal polyps or colorectal cancer for whom blood samples were collected and aliquoted in 7 cryotubes coated with heparin for plasma preparation. Furthermore, blood samples were collected from the same patients for 2 more consecutive days (3 days in total) in order to evaluate whether fluctuations of the cell-free DNA level could happen in the bloodstream over a period of time. Plasma separation and DNA extraction were performed as previously described.6 For the first set of experiments, which were focused on the evaluation of between-measurement reproducibility and within-subject variability, samples obtained on day 1 were divided into two aliquots; one was immediately processed (detA), the second was stored at –20°C until analysis, which occurred 3 months later (detB). For both samples, DNA quantification was repeated blindly after 2 weeks; thus for detA and detB two independent measurements were available, with a total of four runs for each of the seven aliquots per subject, and an overall total of 420 measurements. All measurements were performed jointly by two operators using the DNA DipStick kit (Invitrogen, Carlsbad, CA).6 For the semiquantitative nature of the measurement, crude DNA values were first categorized into six classes,6 and each comparison agreement was expressed by weighted kappa statistic (kw).7 Once operator performance was assessed (kw value ranges, 0.939-1.00), our interests were: (1) to verify the reproducibility between measurements obtained independently in two distinct determinations and (2) to evaluate the within-subject classification variability. A satisfactory level of reproducibility was observed within/between determinations (kw value ranges, 0.867-0.949), and absolute DNA content values were concordant in detA and detB after a 3-month delay in quantification. Overall, regarding the within-subject classification variability, agreement in DNA levels was observed in 358 of 420 instances (85% of the measurements); concordance among measurements within subjects was complete for two patients, greater than 90% or between 90% and 80% for four and five patients, respectively. In four patients, disagreements were present in 10 to 12 of the 28 measurements, but limited to a single-class shift. Once it was assessed that the DNA DipStick kit is a reliable diagnostic device able to quantify rapidly and reproducibly free-circulating DNA in plasma, we determined cell-free DNA levels over 3 consecutive days in the same patients and we found complete measurement concordance in 14 patients whereas the crude absolute values were quite similar but borderline in one patient. Our results indicate that using a blood-processing protocol similar to that previously reported,4 which does not affect the day-to-day variation in total DNA quantification in maternal plasma, cell-free DNA levels appear to be superimposable over a period of time. This finding demonstrates that cell-free DNA level observed in cancer patient follow-up is a stable feature, also overtime and its variation could be attributed to actual changes in patient outcome. Such data support investigations on the role of cell-free DNA as a potential noninvasive tool for early diagnosis of relapse or metastasis in specific tumor types. Authors' Disclosures of Potential Conflicts of Interest The authors indicated no potential conflicts of interest. REFERENCES
1. Bunn PJ: Early detection of lung cancer using serum RNA or DNA markers: Ready for "prime time" or for validation? J Clin Oncol 21:3891-3893, 2003
2. Sozzi G, Conte D, Leon ME, et al: Quantification of free-circulating DNA as a diagnostic marker in lung cancer. J Clin Oncol 21:3902-3908, 2003
3. Jung M, Klotzek S, Lewandowski M, et al: Changes in concentration of DNA in serum and plasma during storage of blood samples. Clin Chem 49:1028-1029, 2003
4. Chiu RWK, Poon LLM, Lau TK, et al: Effects of blood-processing protocols on fetal and total DNA quantification in maternal plasma. Clin Chem 47:1607-1613, 2001 5. Ziegler A, Zangemeister-Wittke U, Stahel RA: Circulating DNA: A new diagnostic gold mine? Cancer Treat Rev 28:255-271, 2002[CrossRef][Medline]
6. Sozzi G, Conte D, Mariani L, et al: Analysis of circulating tumor DNA in plasma at diagnosis and during follow-up of lung cancer patients. Cancer Res 61:4675-4678, 2001 7. Corletto V, Verderio P, Giardini R, et al: Evaluation of residual cellularity and proliferation on preoperatively treated breast cancer: A comparison between image analysis and light microscopic analysis. Anal Cell Pathol 16:83-93, 1998[Medline]
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Copyright © 2005 by the American Society of Clinical Oncology, Online ISSN: 1527-7755. Print ISSN: 0732-183X
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