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1 1896 Chinese Journal of Pathophysiology IME - FICC * IME - FICC CTCs 76 EpCAM CK 8 / CK8 /18 63 CK8 / % 63 /76 P < CK8 /18 + CTC P < VCA - IgA P > CK8 /18 + CTCs 3 VCA - IgA CK8 /18 + CTCs R A doi /j. issn 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 China 2 Department of Medical Oncology The First Affiliated Hospital of Guangdong Pharmaceutical University Guangzhou China 3 Department of Urinary Surgery The Second Affiliated Hospital Sun Yat - sen University Guangzhou 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 < Relapse patients had significantly higher number of median CK8 /18 + CTCs than the patients without relapse P < No association of viral capsid antigen VCA - IgA P > 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 < * No No. 2011B Tel E - mail huanghq@ sysucc. org. cn

2 when the numbers of peripheral blood CK8 /18 + CTCs were 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 > when the numbers of peripheral blood CK8 /18 + CTCs were 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 % Sex Male NPC Female Age year > T stage T AJCC T circulating tumor cells CTCs 1 - T3 T N stage N immunomagnetic enrichment and fluorescent immunocytochemistry 2002 AJCC N IME - FICC N N CTCs Overall stage Ⅰ IME - FICC Ⅱ NPC CTCs Ⅲ Gy / / 3 1 / + 7 EpiSep 20 cytokeratin CK 8 /18 ELF - 97 DAPI CNE1 Ⅳ Performance status ECOG Failure site s Nasopharynx /neck Distant Nasopharynx /neck + distant I - IVb AJCC American Joint Committee on Cancer ECOG Eastern 2002 AJCC American Joint Committee on Cooperative Oncology Group. Cancer CNE1 WHO II /III I II - IVb ATCC 0. 25% DMEM 2-3 / Life Technologies Histopaque Sigma Gy / Gy /6-7 OncoQuest PBMC

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.

3 Beckman 37 5% CO 2 CK8 / CTCs VCA - IgA Wilcoxon CK8 /18 + CTCs 2 2 Fisher VCA - IgA < ml 2 2 CK 8 /18 + CTCs VCA - IgA Histopaque 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 CK8 /18 + DAPI DNA CNE CK8 / % 20 CK8 / CK8 /18 + P < W 100% 20 /20 Nikon DAPI ELF VCA - IgA DAPI CTCs ELF P < 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 ** No relapse VCA viral capsid antigen. 5 ** P < vs no relapse. SPSS Kaplan - Meier log - rank 63

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.

4 % 13 CK + 1 P > VCA - IgA % 3 /12 VCA - IgA < % 14 /64 P > % % 100% 94. 1% 71. 4% 53. 3% 44. 4% P < % 100% 100% 100% 100% 80. 0% 77. 8% P > VCA - IgA < % 80. 6% P > Figure % 94. 0% P > Cox CTCs Relapse - free survival A and overall survival B with different numbers of CTCs. P < CTCs Cox CTCs P < hazard ratio HR = % confidence interval CI = 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

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.

5 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 < < Response PR/SD vs CR > < Overall stage III - IV vs I - II > < Age 50 vs > 50 > Not relevant in Cox model Sex male vs female > Not relevant in Cox model PS 0 vs 1 > Not relevant in Cox model T 1-2 vs 3-4 > Not relevant in Cox model N 0 vs 1-3 > Not relevant in Cox model VCA - IgA 1 80 vs < 1 80 > Not relevant in Cox model % CI = 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 < < Age 50 vs > 50 < Response PR/SD vs CR < Not relevant in Cox model Overall stage III - IV vs I - II < Not relevant in Cox model Sex male vs female > Not relevant in Cox model PS 0 vs 1 > Not relevant in Cox model T 1-2 vs 3-4 > Not relevant in Cox model N 0 vs 1-3 > Not relevant in Cox model VCA - IgA 1 80 vs < 1 80 > Not relevant in Cox model immunocytochemistry ICC PCR ICC CK CK 9 10 ICC EpiSep % CK8 /18 + CTCs P < P < P < P < CTCs 6 7 CK8 /18 Cox + CTCs IME - FICC P < HR = % CI = P = 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 % CTCs FDG - PET /CT CTCs FDG - PET /CT P < P < CTCs % FDG - PET /CT P <

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.

6 CTC analysis of cancer cells in blood and bone marrow using a P < rare event imagingsystem J. Clin Cancer Res 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 Hu XC Wang Y Shi DR et al. Immunomagnetic tumor 64 cell enrichment is promising in detecting circulating breast % 5 4 Cristofanilli M Broglio KR Guarneri V et al. Circulating tumor cells in metastatic breast cancer biologic staging P < cancer cells J. Oncology beyond tumor burden J. Clin Breast Cancer Paterlini - Brechot P Benali NL. Circulating tumor cells CTC detection Clinical impact and future directions J. Cancer Lett P < J IME - FICC J J CK8 / CK - 20 J CK19 J 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 VCA - IgA Okegawa T Nutahara K Higashihara E. Prognostic significance of circulating tumor cells in patients with hormone refractory prostate cancer J. J Urol Kraeft SK Sutherland R Gravelin L et al. Detectionand

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.

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