Method validation of circulating tumour cell enumeration at low cell counts

BMC Cancer

Jeffrey Cummings¹  Karen Morris¹  Cong Zhou¹  Robert Sloane¹  Matt Lancashire¹  Daniel Morris¹  Stephen Bramley¹  Matt Krebs¹  Leila Khoja¹  Caroline Dive¹ 

[1] Clinical and Experimental Pharmacology Group, Paterson Institute for Cancer Research, Manchester Cancer Research Centre, University of Manchester, Manchester M20 4BX, UK

关键词: Statistical analysis;    Method validation;    Low cell counts;    Enumeration;    Predictive biomarker;    Circulating tumour cells;   

Others  :  1079575
DOI  :  10.1186/1471-2407-13-415

 received in 2013-05-31, accepted in 2013-09-02,  发布年份 2013

【 摘 要 】

Background

Circulating tumour cells (CTC) are receiving increasing attention as prognostic, predictive and pharmacodynamic biomarkers in cancer patients. However, their clinical significance can be dependent on an accurate determination of CTC around cut-off values at low cell counts (<10 cells/7.5 ml). Consequently, we have conducted method validation of the CellSearch™ system focusing on clinical samples containing CTC in the cut-off region.

Methods

Analytical accuracy was first assessed employing quality controls (QC) and spiked healthy volunteer blood specimens. Results were analysed by β-expectation tolerance intervals (BETI). Inter-operator error (6 different readers) was then characterised in 38 different patient samples, 68% of which had ≤5 CTC and data were analysed by β-content γ-confidence tolerance intervals (BCTI).

Results

Results from QCs and spiked blood confirmed a 3-4-fold higher degree of imprecision at the low (48 cells, BETI = + 0.288/-0.345, β = 95%) compared to the high QC (987 cells, BETI = +0.065/-0.140, β = 95%). However, when data for individual analysts were interrogated characteristic systematic errors were detected. In the analysis of patient samples again individual analysts introduced a highly specific error into the interpretation of CTC images, which correlated to the level of training and experience. When readers were selected based on BETI and BCTI results, the high level of between-operator error (up to 170%) observed at CTC of ≤ 5 was reduced to < 30%.

Conclusions

Inter-operator variability in enumeration of CTC at low cell counts can be considerable, but is also potentially avoidable by following simple guidance steps.

【 授权许可】

   
2013 Cummings et al.; licensee BioMed Central Ltd.

【 图 表 】

Figure 1.

Figure 2.

Figure 3.

【 参考文献 】

• [1]Alix-Panabieres C, Pantel K: Circulating tumor cells: liquid biopsy of cancer. Clin Chem 2013, 59(1):110-118.
• [2]Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Matera J, Miller MC, Reuben JM, Doyle GV, Allard WJ, Terstappen LW, et al.: Circulating tumor cells, disease progression, and survival in metastatic breast cancer. N Engl J Med 2004, 351(8):781-791.
• [3]Cohen SJ, Punt CJ, Iannotti N, Saidman BH, Sabbath KD, Gabrail NY, Picus J, Morse M, Mitchell E, Miller MC, et al.: Relationship of circulating tumor cells to tumor response, progression-free survival, and overall survival in patients with metastatic colorectal cancer. J Clin Oncol 2008, 26(19):3213-3221.
• [4]Danila DC, Heller G, Gignac GA, Gonzalez-Espinoza R, Anand A, Tanaka E, Lilja H, Schwartz L, Larson S, Fleisher M, et al.: Circulating tumor cell number and prognosis in progressive castration-resistant prostate cancer. Clin Cancer Res 2007, 13(23):7053-7058.
• [5]Kim MY, Oskarsson T, Acharyya S, Nguyen DX, Zhang XH, Norton L, Massague J: Tumor self-seeding by circulating cancer cells. Cell 2009, 139(7):1315-1326.
• [6]Krebs MG, Sloane R, Priest L, Lancashire L, Hou JM, Greystoke A, Ward TH, Ferraldeschi R, Hughes A, Clack G, et al.: Evaluation and prognostic significance of circulating tumor cells in patients with non-small-cell lung cancer. J Clin Oncol 2011, 29(12):1556-1563.
• [7]Khoja L, Lorigan P, Zhou C, Lancashire M, Booth J, Cummings J, Califano R, Clack G, Hughes A, Dive C: Biomarker utility of circulating tumor cells in metastatic cutaneous melanoma. J Invest Dermatol 2013, 133(6):1582-1590.
• [8]Khan MS, Kirkwood A, Tsigani T, Garcia-Hernandez J, Hartley JA, Caplin ME, Meyer T: Circulating tumor cells as prognostic markers in neuroendocrine tumors. J Clin Oncol 2013, 31(3):365-372.
• [9]Tol J, Koopman M, Miller MC, Tibbe A, Cats A, Creemers GJ, Vos AH, Nagtegaal ID, Terstappen LW, Punt CJ: Circulating tumour cells early predict progression-free and overall survival in advanced colorectal cancer patients treated with chemotherapy and targeted agents. Ann Oncol 2010, 21(5):1006-1012.
• [10]De-Bono JS, Scher HI, Montgomery RB, Parker C, Miller MC, Tissing H, Doyle GV, Terstappen LW, Pienta KJ, Raghavan D: Circulating tumor cells predict survival benefit from treatment in metastatic castration-resistant prostate cancer. Clin Cancer Res 2008, 14(19):6302-6309.
• [11]Scher HI, Jia X, De-Bono JS, Fleisher M, Pienta KJ, Raghavan D, Heller G: Circulating tumour cells as prognostic markers in progressive, castration-resistant prostate cancer: a reanalysis of IMMC38 trial data. Lancet Oncol 2009, 10(3):233-239.
• [12]Devriese LA, Voest EE, Beijnen JH, Schellens JH: Circulating tumor cells as pharmacodynamic biomarker in early clinical oncological trials. Cancer Treat Rev 2011, 37(8):579-589.
• [13]Mostert B, Sleijfer S, Foekens JA, Gratama JW: Circulating tumor cells (CTCs): detection methods and their clinical relevance in breast cancer. Cancer Treat Rev 2009, 35(5):463-474.
• [14]Tibbe AG, De-Grooth BG, Greve J, Dolan GJ, Terstappen LW: Imaging technique implemented in Cell Tracks system. Cytometry 2002, 47(4):248-255.
• [15]Riethdorf S, Fritsche H, Muller V, Rau T, Schindlbeck C, Rack B, Janni W, Coith C, Beck K, Janicke F, et al.: Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the Cell Search system. Clin Cancer Res 2007, 13(3):920-928.
• [16]Allard WJ, Matera J, Miller MC, Repollet M, Connelly MC, Rao C, Tibbe AG, Uhr JW, Terstappen LW: Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res 2004, 10(20):6897-6904.
• [17]Cummings J, Ward TH, Greystoke A, Ranson M, Dive C: Biomarker method validation in anticancer drug development. Br J Pharmacol 2008, 153(4):646-656.
• [18]Parkinson DR, Dracopoli N, Petty BG, Compton C, Cristofanilli M, Deisseroth A, Hayes DF, Kapke G, Kumar P, Lee J, et al.: Considerations in the development of circulating tumor cell technology for clinical use. J Transl Med 2012, 10:138. BioMed Central Full Text
• [19]Khleif SN, Doroshow JH, Hait WN: AACR-FDA-NCI Cancer Biomarkers Collaborative consensus report: advancing the use of biomarkers in cancer drug development. Clin Cancer Res 2010, 16(13):3299-3318.
• [20]Dancey JE, Dobbin KK, Groshen S, Jessup JM, Hruszkewycz AH, Koehler M, Parchment R, Ratain MJ, Shankar LK, Stadler WM, et al.: Guidelines for the development and incorporation of biomarker studies in early clinical trials of novel agents. Clin Cancer Res 2010, 16(6):1745-1755.
• [21]Kraan J, Sleijfer S, Strijbos MH, Ignatiadis M, Peeters D, Pierga JY, Farace F, Riethdorf S, Fehm T, Zorzino L, et al.: External quality assurance of circulating tumor cell enumeration using the Cell Search((R)) system: a feasibility study. Cytometry B Clin Cytom 2011, 80(2):112-118.
• [22]Rozet E, Ceccato A, Hubert C, Ziemons E, Oprean R, Rudaz S, Boulanger B, Hubert P: Analysis of recent pharmaceutical regulatory documents on analytical method validation. J Chromatogr A 2007, 1158(1–2):111-125.
• [23]Hoffman D: Statistical considerations for assessment of bioanalytical incurred sample reproducibility. AAPS J 2009, 11(3):570-580.
• [24]Tibbe AG, Miller MC, Terstappen LW: Statistical considerations for enumeration of circulating tumor cells. Cytometry A 2007, 71(3):154-162.
• [25]Hou JM, Greystoke A, Lancashire L, Cummings J, Ward T, Board R, Amir E, Hughes S, Krebs M, Hughes A, et al.: Evaluation of circulating tumor cells and serological cell death biomarkers in small cell lung cancer patients undergoing chemotherapy. Am J Pathol 2009, 175(2):808-816.
• [26]Cummings J, Zhou C, Dive C: Application of the beta-expectation tolerance interval to method validation of the M30 and M65 ELISA cell death biomarker assays. J Chromatogr B Analyt Technol Biomed Life Sci 2011, 879(13–14):887-893.
• [27]Rao C, Bui T, Connelly M, Doyle G, Karydis I, Middleton MR, Clack G, Malone M, Coumans FA, Terstappen LW: Circulating melanoma cells and survival in metastatic melanoma. Int J Oncol 2011, 38(3):755-760.
• [28]Allan AL, Keeney M: Circulating tumor cell analysis: technical and statistical considerations for application to the clinic. J Oncol 2010, 2010:426218.
• [29]Coumans FA, Ligthart ST, Uhr JW, Terstappen LW: Challenges in the enumeration and phenotyping of CTC. Clin Cancer Res 2012, 18(20):5711-5718.
• [30]Feinberg M: Validation of analytical methods based on accuracy profiles. J Chromatogr A 2007, 1158(1–2):174-183.
• [31]Boemer F, Bours V, Schoos R, Hubert P, Rozet E: Analytical validation based on total error measurement and cut-off interpretation of a neonatal screening TSH-immunoassay. J Chromatogr B Analyt Technol Biomed Life Sci 2009, 877(23):2412-2417.
• [32]Feinberg M, Boulanger B, Dewe W, Hubert P: New advances in method validation and measurement uncertainty aimed at improving the quality of chemical data. Anal Bioanal Chem 2004, 380(3):502-514.
• [33]Lee JW, Devanarayan V, Barrett YC, Weiner R, Allinson J, Fountain S, Keller S, Weinryb I, Green M, Duan L, et al.: Fit-for-purpose method development and validation for successful biomarker measurement. Pharm Res 2006, 23(2):312-328.
• [34]Lee JW, Weiner RS, Sailstad JM, Bowsher RR, Knuth DW, O’Brien PJ, Fourcroy JL, Dixit R, Pandite L, Pietrusko RG, et al.: Method validation and measurement of biomarkers in nonclinical and clinical samples in drug development: a conference report. Pharm Res 2005, 22(4):499-511.
• [35]Findlay JW: Some important considerations for validation of ligand-binding assays. J Chromatogr B Analyt Technol Biomed Life Sci 2009, 877(23):2191-2197.
• [36]Cummings J, Raynaud F, Jones L, Sugar R, Dive C: Fit-for-purpose biomarker method validation for application in clinical trials of anticancer drugs. Br J Cancer 2010, 103(9):1313-1317.
• [37]Jennings L, Van-Deerlin VM, Gulley ML: Recommended principles and practices for validating clinical molecular pathology tests. Arch Pathol Lab Med 2009, 133(5):743-755.
• [38]Scher HI, Morris MJ, Larson S, Heller G: Validation and clinical utility of prostate cancer biomarkers. Nat Rev Clin Oncol 2013, 10(4):225-234.
• [39]Hougton R, Gouty D, Allinson J, Green R, Losauro M, Lowes S, LeLacheur R, Garofolo F, Couerbe P, Bronner S, et al.: Recommendations on biomarker bioanalytical method validation by GCC. Biogeosciences 2012, 4(20):2439-2446.
• [40]Hayashi N, Yamauchi H: Role of circulating tumor cells and disseminated tumor cells in primary breast cancer. Breast Cancer 2012, 19(2):110-117.