Circulating tumor cells: a valuable marker of poor prognosis for advanced nasopharyngeal carcinoma
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To evaluate the prognostic value of circulating tumor cells (CTCs) in nasopharyngeal carcinoma (NPC).
Cox’s proportional hazards regression models were used to identify whether CTCs was a poor prognostic factor for NPC. Chi-square tests were used to analyze and compare the distribution characteristics of CTCs in NPC. ROC curve was used to estimate the cut-off point of CTCs. Kaplan-Meier survival analyses were used to observe the prognostic value of CTCs alone and in combined with Epstein-Barr Virus DNA (EBV-DNA).
CTCs was confirmed to be an independent risk factor for poor prognosis of NPC by Cox’s regression models that enrolled 370 NPC cases and took age, gender, EBV-DNA and CTCs as variables. The proportion of CTCs in stage IV NPC was statistically different from that in stage III; the cut-off point of CTCs between stage IV (288 cases) and stage III (70 cases) NPC estimated by ROC curve was 0.5. The prognosis of advanced NPC patients became worse with the increase of CTCs count. The combined detection of CTCs and EBV-DNA could better predict the prognosis of NPC compared with the single detection of EBV-DNA.
KeywordsNasopharyngeal carcinoma Circulating tumor cells Epstein-Barr virus DNA Prognosis Cut-off point
Circulating tumor cells
Epstein-Barr Virus DNA
Circulating tumor cells (CTCs) count using the CellSearch system (Janssen Diagnostics, Raritan, NJ, USA) has been proved to have good prognostic value for metastatic breast cancer (Bidard et al. 2014). Due to expensive cost of CTCs examination and regional characteristics of nasopharyngeal carcinoma (NPC), the application of CTCs in NPC is still in exploratory stage (Si et al. 2016; Zhang et al. 2018; He et al. 2017); Meanwhile, the detection methods of CTCs were also different in different studies. Compared with Epstein-Barr Virus DNA (EBV-DNA), the application value of CTCs in NPC is controversial (He et al. 2017; Vo et al. 2016). The reasons for different conclusions may be that the detection methods of CTCs or the research approaches adopted by different studies were different, or there were racial differences among patients enrolled in different studies. In this study, paired data of CTCs and EBV-DNA were analyzed by Kaplan-Meier survival analyses to confirm the role of CTCs in the prognosis of NPC. This study focused on whether CTCs has prognostic value for NPC and how to use CTCs to predict the prognosis of NPC patients. It should be noted that the patients enrolled in this study were mainly from southern China.
Instrument and reagent
Instrument: CellTracks® AutoPrep® system and CellTracks® Analyzer II.
Manufacturer: Janssen Diagnostics, LLC.
Kit name: CellSearch Circulating Tumor Cell Kit (Epithelial).
Detection principle: The detection kit is based on ferromagnetic fluid capture reagent and immunofluorescence reagent; the ferromagnetic fluid reagent contains nanoparticles with magnetic cores, the polymer layer around the magnetic core is coated with antibodies against EpCAM antigens to capture CTCs. After the CTCs has been captured and enriched by the immune magnet, fluorescent reagents are added to identify and count CTCs. The fluorescence reagent contains the following components: anti-CK-phycoerythrin (PE) specific to cytokeratin (characteristic of epithelial cells), DAPI for nuclear staining and anti-CD45-phycocyanin (APC) specific to leukocyte.
CellTracks® Analyzer II automatically scan the entire surface of the sample box, capture image and present any fluorescent glow event by CK-PE together with DAPI to the user. If its morphological characteristics are consistent with those of tumor cells, and the immune typing is shown as EpCAM+, CK+, DAPI+, and CD45-, it will be classified as tumor cells.
Limitations of the method: If the patient is receiving doxorubicin, the blood should not be drawn until at least 7 days after doxorubicin injection. If a blood sample is taken within 7 days of doxorubicin administration, the interpretation of CellSearch® test results must be cautious. CellSearch® can’t detect CTCs without expression of EpCAM or cytokeratin-8, 18 or 19.
Interpretation of test results: The reported test result was <CTCs count>/7.5 ml of blood.
The cases data enrolled in this study were from primary or recurrent tumor patients who visited our hospital from August 2015 to March 2018. All patients who underwent CTCs examination during this period were analyzed in this retrospective study, including patients with NPC or other tumor types. Clinicians mainly considered the patients’ clinical stages when applying for CTCs examination, and were more inclined to perform CTCs assessment on patients with advanced cancer. The moment of CTCs assessment during the treatment varies, from 2981 days after to 39 days before the first treatment, details of the assessment moment were shown in Additional file 1: Figure S1. Treatments before or after CTCs assessment mainly included chemotherapy and radiotherapy; Some patients with distant metastases would undergo local surgery for the site of metastases, such as radiofrequency ablation, vertebroplasty and lymph node dissection; A few patients participated in clinical trials of new drugs such as triprizumab. Since NPC patients were mainly treated with chemotherapy and radiotherapy, and the proportion of patients receiving surgical treatment was relatively low, there was no statistical analysis on surgical treatment in Additional file 1: Figure S1.
For NPC, there were 307 males, aged 15–80 yr, the median age was 45; and 66 females, aged 15–71 yr, the median age was 43. The proportions of NPC patients in each clinical stage were showed in Additional file 2: Table S1.
The copy of EBV-DNA was detected by Cobas Z 480 fluorescence quantitative PCR instrument (The reference value was < 103 copies/ml). Sample collection, processing and detection of CTCs and EBV-DNA were carried out according to the instructions provided by the manufacturer. The staging of NPC and other tumor types analyzed in this study was referred to AJCC tumor staging manual, 7th edition.
Overall survival was defined as time from baseline CTCs assessment to death from any cause. Patients without documented evidence of death were censored at the date of last follow-up. Chi-square tests were used to analyze the proportions of CTCs in different clinical stages, age groups and genders. ROC curve analysis was used to estimate the cut-off point of CTCs between stage III and stage IV NPC. Cox’s regression models combined with Kaplan-Meier survival analyses were used to confirm the relationship between elevated CTCs count and poor prognosis of NPC.
CTCs is an independent risk factor for poor prognosis of NPC
Variables assignment and analysis results of Cox’s regression models for NPC
Grouping and assignment
95.0% CI for HRs
1 = “male”; 2 = “female”
1 = “I”; 2 = “II”; 3 = “III”; 4 = “IV”
1 = “male”; 2 = “female”
1 = “I”; 2 = “II”; 3 = “III”; 4 = “IV”
The distribution characteristics of CTCs in NPC
Proportions of CTCs in each NPC clinical stage and their comparison
Number of cases
Total number of cases
P-value of Chi-square Test:
Prognostic value observation of CTCs
In view of the factors that affecting the prognosis of NPC also include age and gender (Xiao et al. 2013; Proceedings of the 7th Biannual International Symposium on Nasopharyngeal Carcinoma 2015 n.d.), the proportions of CTCs in different age groups and genders were also analyzed here (Additional file 4: Table S3 and Additional file 5: Table S4). As can be seen from Additional file 4: Table S3, although the prognosis of male NPC patients was worse than that of female (OuYang et al. 2015), there was no statistical difference in the proportions of CTCs between male and female. From Additional file 5: Table S4, although the difference was not significant, it seemed that tumor cells of elderly advanced NPC patients were more likely to be shed from the primary site into the bloodstream, resulting in increased CTCs count; As opposed to that, there was no statistical difference in the proportions of CTCs between the two age groups (<45y and > =45y) in stage III NPC, suggesting that CTCs did not increase with age in stage III NPC.
Combined detection of CTCs and EBV-DNA can better predict the prognosis of NPC
The role of EBV-DNA in the prognosis of NPC has been confirmed (Liu et al. 2017; Prayongrat et al. 2017). The cut-off point of EBV-DNA vary among different studies, such as 8000 copies/ml or 2300 copies/ml (Chai et al. 2012; Lertbutsayanukul et al. 2018). The patients enrolled in the Kaplan-Meier survival analyses in this subparagraph were grouped by EBV-DNA = 4000 copies/ml (Chen et al. 2016) and CTCs = 1/7.5 ml. To avoid the interference of selection bias, Kaplan-Meier analyses in this subparagraph were only performed on paired EBV-DNA and CTCs data. Paired data of EBV-DNA and CTCs was obtained from blood samples taken from the same patient within 3 days, mostly from the same blood sampling.
A study from Singapore showed that there was no statistically significant relationship between CTCs and clinical stages (Vo et al. 2016). However, data analyses in this study showed that there was a statistical difference between the proportions of CTCs in stage III and stage IV NPC (Table 2). The study from Singapore used a technology named Microsieve to detect CTCs (Lim et al. 2012). The Microsieve technology was a size-based method capable of isolating both epithelial and mesenchymal CTCs, utilizing the size and deformability differences between the CTCs and normal blood cells; In contrast, Cellsearch system captured CTCs by nanoparticles with magnetic cores which were coated with antibodies against EpCAM antigens. The fundamental difference between Microsieve and Cellsearch was the way CTCs were captured, that was, size-based or EpCAM-based. Besides, the definition of CTCs was not quite the same between Microsieve and Cellsearch; Microsieve classified CK+, CD45- nucleated cells as canonical CTCs, while Cellsearch classified EpCAM+, CK+, DAPI+, and CD45- nucleated cells as tumor cells. In our opinion, the most important reason for the two studies to reach different conclusions was the different ways of capturing CTCs.
Although Cox’s regression models in subparagraph 1 and Kaplan-Meier survival analyses in subparagraph 4 analyzed all available cases collected by this study, regardless of gender and clinical stage, most of the cases analyzed in these two subparagraphs were male patients with stage IV NPC. Therefore, the conclusions of this study are best applicable to male advanced NPC.
In the multivariate Cox’s regression model, only 64 patients (20.5%) were followed up to the end event (Death), this may affect the accuracy of statistical results. Since the survival time of NPC patients is generally long, this problem is inevitable now. This exploratory study suggests that CTCs is a possible prognostic biomarker. But its definitive evaluation, above all, its comparison to EBV-DNA, should be necessarily evaluated in a prospective controlled study, with more focused eligibility criteria.
Cox’s regression models confirmed that CTCs was an independent risk factor affecting the prognosis of NPC. By analyzing the distribution characteristics of CTCs in NPC and comparing the differences of different CTCs counts in predicting the prognosis of advanced NPC patients, it was confirmed that the prognosis of advanced NPC patients became worse with the increase of CTCs count. Kaplan-Meier survival analyses confirmed that the combined detection of CTCs and EBV-DNA could better predict the prognosis of NPC.
GPO: involved in protocol development, data analyses and wrote the first draft of the manuscript; SLC: researched literature, conceived the study and reviewed patients’ information; All authors reviewed and edited the manuscript and approved the final version of the manuscript.
Ethics approval and consent to participate
The only record of contacting subjects for identification and research is the informed consent document, signed informed consent poses an undue threat to the subject’s privacy. This study is based on retrospective analysis, exemption from ethical approval and informed consent will not adversely affect the subject’s rights and health. Therefore, this study is exempt from ethical approval and informed consent.
Consent for publication
The authors declare that they have no competing interests.
- Yogyakarta, Indonesia. Proceedings of the 7th Biannual International Symposium on Nasopharyngeal Carcinoma 2015. BMC Proceedings. 2016;10(Suppl 1).Google Scholar
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