1896 Chinese Journal of Pathophysiology 2011 27 10 1896-1901 1000-4718 2011 10-1896 - 06 IME - FICC * 1 2 3 1 1 510060 2 510080 3 510120 IME - FICC CTCs 76 EpCAM CK 8 /18 25 20 CK8 /18 63 CK8 /18 82. 9% 63 /76 P < 0. 01 CK8 /18 + CTC P < 0. 01 VCA - IgA P > 0. 05 CK8 /18 + CTCs 3 VCA - IgA CK8 /18 + CTCs R739. 62 A doi 10. 3969 /j. issn. 1000-4718. 2011. 10. 008 Detection and clinical significance of circulating tumor cells in peripheral blood of nasopharyngeal cancer patients with modified immunomagnetic enrichment and fluorescent immunocytochemistry CAI Qing - qing 1 DING Ying 2 LIN Tian - xin 3 HUANG Hui - qiang 1 1 Department of Medical Oncology Cancer Center Sun Yat - sen University Guangzhou 510060 China 2 Department of Medical Oncology The First Affiliated Hospital of Guangdong Pharmaceutical University Guangzhou 510080 China 3 Department of Urinary Surgery The Second Affiliated Hospital Sun Yat - sen University Guangzhou 510120 China. E - mail huanghq@ sysucc. org. cn ABSTRACT AIM To investigate the clinical significance of the new method of modified immunomagnetic enrichment of tumor cells in combination with fluorescent immunocytochemistry to detect circulating tumor cells CTCs in peripheral blood samples of patients with nasopharyngeal cancer NPC. METHODS Peripheral blood samples were obtained from 76 histology - proven patients with NPC before the initial therapy. After isolation of the mononuclear cells the CTCs expressing cytokeratin CK 8 /18 in the blood samples were detected by the method of immunomagnetic enrichment in combination with fluorescent immunocytochemistry. The magnetic beads covalent binding with epithelial cell adhesion molecule EpCAM antibody were used to enrich the tumor cells which expressed EpCAM. After median following - up for 25 months the effects of CTCs and other prognostic factors on patient prognosis were thoroughly investigated. RESULTS None of the positive CK8 /18 cells was detected in 20 normal blood samples. The CTCs were detected in 82. 9% of the patients P < 0. 01. Relapse patients had significantly higher number of median CK8 /18 + CTCs than the patients without relapse P < 0. 01. No association of viral capsid antigen VCA - IgA P > 0. 05 was observed between the patients with and without relapse. Relapse - free survival rates were lower when the number of peripheral blood CK8 /18 + CTCs was more than 3. The 2 - year relapse - free survival rates were 100% 100% 100% 94. 1% 71. 4% 53. 3% and 44. 4% P < 2011-06 - 17 * 2011-09 - 01 No. 81001052 No. 2011B031800222 Tel 020-87343350 E - mail huanghq@ sysucc. org. cn
1897 0. 01 when the numbers of peripheral blood CK8 /18 + CTCs were 0 1 2 3 4 5 6 before treatment respectively. Overall survival rates were lower when the number of peripheral blood CK8 /18 + CTCs was more than 5 but the difference was not significant. The 2 - year overall survival rates were 100% 100% 100% 100% 100% 100% 80% and 77. 8% P > 0. 05 when the numbers of peripheral blood CK8 /18 + CTCs were 0 1 2 3 4 5 6 before treatment respectively. The VCA - IgA titer could not predict survivals. Cox multivariate analysis also showed the same results. CON- CLUSION Peripheral blood CK8 /18 + CTCs are a prognostic factor for initial treatment of NPC. KEY WORDS Nasopharyngeal neoplasms Circulating tumor cells Immunomagnetic enrichment Immunocytochemistry nasopharyngeal carcinoma Table 1. Clinical characteristics of NPC patients n = 76 NPC 1 76 Item n % 5 000 Sex Male 62 81. 6 3 000 NPC Female 14 18. 4 Age year 50 51 67. 1 > 50 25 32. 9 T stage T1 11 14. 5 2002 AJCC T2 24 31. 6 circulating tumor cells CTCs 1 - T3 T4 30 39. 5 11 14. 5 N stage N0 17 22. 4 immunomagnetic enrichment and fluorescent immunocytochemistry 2002 AJCC N1 41 53. 9 IME - FICC N2 16 21. 1 N3 2 2. 6 CTCs Overall stage Ⅰ 3 3. 9 6 IME - FICC Ⅱ 28 36. 8 NPC CTCs Ⅲ 32 42. 1 1 50-56 Gy /5-5. 5 1 25 5-35 17 13 / 3 1 / + 7 EpiSep 20 cytokeratin CK 8 /18 ELF - 97 DAPI CNE1 Ⅳ 13 17. 1 Performance status 0 9 11. 8 ECOG 1 67 88. 2 2008 1-2008 12 76 Failure site s Nasopharynx /neck 13 17. 1 Distant 3 3. 9 Nasopharynx /neck + distant 1 1. 3 I - IVb AJCC American Joint Committee on Cancer ECOG Eastern 2002 AJCC American Joint Committee on Cooperative Oncology Group. Cancer 76 2 2. 1 CNE1 WHO II /III I II - IVb ATCC 0. 25% DMEM 2-3 / Life Technologies Histopaque - 1077 Sigma 66-70 Gy /6. 5-7 60-70 Gy /6-7 OncoQuest PBMC
1898 2. 2 Beckman 37 5% CO 2 CK8 /18 1 8 CTCs VCA - IgA Wilcoxon CK8 /18 + CTCs 2 2 Fisher 6 7 3 VCA - IgA < 1 80 1 80 2 3. 1 15 ml 2 2 CK 8 /18 + CTCs VCA - IgA Histopaque - 1077 Gehan Cox epidermal cellular adhesion molecule EpCAM Forward LR EpCAM 50 μl 950 μl EpiSep 1 76 I - IVb 1 CK8 /18 + EpCAM CK8 /18 CK8 /18 dsdna DA- - PI ELF - 97 2 CK8 /18 + DAPI DNA CNE1 76 63 CK8 /18 + 82. 9% 20 CK8 /18 + 3. 2 CK8 /18 + P < 0. 01 100W 100% 20 /20 Nikon DAPI ELF97 3 2 VCA - IgA DAPI CTCs ELF97 5 5-6 3 1-4 P < 0. 01 CK8 /18 VCA - IgA 2 SPOT Advanced 3. 4 for Windows Diagnostic Instruments - IgA 2 CK8 /18 + VCA Table 2. CK8 /18 + circulating tumor cells CTCs and VCA - 4 viral capsid antigen VCA - IgA antibody titers before treatment in the patients with IgA n = 17 or without n = 59 relapse VCA - IgA CK8 /18 + CTCs VCA - IgA PBS 1 5 Median Interquartile range Median Interquartile range Relapse 5 ** 5-6 1 320 1 160-1 320 8 No relapse 3 1-4 1 320 1 160-1 640 VCA viral capsid antigen. 5 ** P < 0. 01 vs no relapse. SPSS 13. 0 4 Kaplan - Meier log - rank 63
1899 17 27. 0% 13 CK + 1 P > 0. 05 VCA - IgA 1 80 25. 0% 3 /12 VCA - IgA < 1 80 21. 9% 14 /64 P > 0. 05 5 2 100% 1 2 3 4 5 6 2 100% 100% 94. 1% 71. 4% 53. 3% 44. 4% P < 0. 01 3 1 2 3 4 5 6 2 100% 100% 100% 100% 100% 80. 0% 77. 8% P > 0. 05 5 2 VCA - IgA < 1 80 1 80 2 77. 8% 80. 6% P > 0. 05 Figure 2. 2 88. 9% 94. 0% P > 0. 05 3 6 Cox CTCs Relapse - free survival A and overall survival B with different numbers of CTCs. P < 0. 01 CTCs Cox CTCs P < 0. 01 hazard ratio HR = 3. 320 95% confidence interval CI = 2. 073-5. 318 3 2 CTCs Figure 1. Circulating tumor cells detected in the peripheral blood of patients with nasopharyngeal cancer 400. A one CK8 /18 + cell was observed with long - pass UV filter B the cell nucleus blue fluorescence stained by DAPI was visible with narrow - pass UV filter. Figure 3. Relapse - free survival A and overall survival B with different levels of serum VCA - IgA titers. 1 3 VCA - IgA
1900 Table 3. 3 Cox Summary of relapse - free survival analyses by multivariate Cox proportional hazards model n = 76 Risk factor Univariate analysis Multivariate analysis P HR HR 95% CI P CK - CTC < 0. 01 3. 320 2. 073-5. 318 < 0. 01 Response PR/SD vs CR > 0. 05 0. 123 0. 015-0. 987 < 0. 05 Overall stage III - IV vs I - II > 0. 05 0. 203 0. 066-0. 623 < 0. 01 Age 50 vs > 50 > 0. 05 Not relevant in Cox model Sex male vs female > 0. 05 Not relevant in Cox model PS 0 vs 1 > 0. 05 Not relevant in Cox model T 1-2 vs 3-4 > 0. 05 Not relevant in Cox model N 0 vs 1-3 > 0. 05 Not relevant in Cox model VCA - IgA 1 80 vs < 1 80 > 0. 05 Not relevant in Cox model 5. 190 95% CI = 1. 001-26. 904 4 Table 4. 4 Cox Summary of overall survival analyses by multivariate Cox proportional hazards model n = 76 Risk factor Univariate analysis Multivariate analysis P HR HR 95% CI P CK - CTC < 0. 01 3. 639 1. 303-10. 158 < 0. 05 Age 50 vs > 50 < 0. 05 5. 190 1. 001-26. 904 0. 05 Response PR/SD vs CR < 0. 05 Not relevant in Cox model Overall stage III - IV vs I - II < 0. 01 Not relevant in Cox model Sex male vs female > 0. 05 Not relevant in Cox model PS 0 vs 1 > 0. 05 Not relevant in Cox model T 1-2 vs 3-4 > 0. 05 Not relevant in Cox model N 0 vs 1-3 > 0. 05 Not relevant in Cox model VCA - IgA 1 80 vs < 1 80 > 0. 05 Not relevant in Cox model immunocytochemistry ICC PCR ICC CK CK 9 10 ICC EpiSep 10 7 1 100% CK8 /18 + CTCs P < 0. 01 P < 0. 05 P < 0. 01 P < 0. 05 CTCs 6 7 CK8 /18 Cox + CTCs IME - FICC P < 0. 05 HR = 3. 639 95% CI = 1. 303-10. 158 P = 0. 050 HR = CTCs VCA - IgA 3 5 VCA - IgA Cox Liu 11 MBC CTCs FDG - PET /CT 9 12 CTCs FDG - PET /CT MBC CTCs < 5 CTCs /7. 5 ml 5 CTCs /7. 5 ml CTCs FDG - PET /CT 102 NPC 14 1 > 41 NPC 102 68 67% CTCs FDG - PET /CT CTCs FDG - PET /CT P < 0. 01 P < 0. 01 34 1-5 CTCs 5 31 91% FDG - PET /CT P <
1901 0. 01 CTC analysis of cancer cells in blood and bone marrow using a P < 0. 01 rare event imagingsystem J. Clin Cancer Res 2000 6 5 CTCs MBC Okegawa 12 2 Witzig TE Bossy B Kimlinger T et al. Detection of circulating cytokeratin - positive cells in the blood of breast CellSearch cancer patients using immunomagnetic enrichment and digital microscopy J. Clin Cancer Res 2002 8 5 1085-1091. 3 Hu XC Wang Y Shi DR et al. Immunomagnetic tumor 64 cell enrichment is promising in detecting circulating breast 64 32 50% 5 4 Cristofanilli M Broglio KR Guarneri V et al. Circulating 13. 0 5 tumor cells in metastatic breast cancer biologic staging 20. 0 P < 0. 01 2 434-442. cancer cells J. Oncology 2003 64 2 160-165. beyond tumor burden J. Clin Breast Cancer 2007 7 6 471-479. 5 Paterlini - Brechot P Benali NL. Circulating tumor cells CTC detection Clinical impact and future directions J. Cancer Lett 2007 253 2 180-204. P < 0. 05 6. J. 2005 24 7 837-841. 7. IME - FICC J. 2010 12 31 76 2005 26 5 596-599. 25 5 35 8. J. 2003 22 1 98 CK8 /18 + - 99. 9. CK - 20 J. 2002 18 8 953-955. 10. CK19 J. 2004 20 5 843-846. 11 Liu MC Shields PG Warren RD et al. Circulating tumor cells a useful predictor of treatment efficacy in metastatic breast cancer J. J Clin Oncol 2009 27 31 5153 - VCA - IgA 5159. 12 Okegawa T Nutahara K Higashihara E. Prognostic significance of circulating tumor cells in patients with hormone refractory prostate cancer J. J Urol 2009 181 3 1091 1 Kraeft SK Sutherland R Gravelin L et al. Detectionand - 1097.