Circulating tumor cells with karyotyping as a novel biomarker for diagnosis and treatment of nasopharyngeal carcinoma

Ethical Committee of West China Hospital of Sichuan University approved the study. Fifty consecutive patients with biopsy-proven nasopharyngeal carcinoma (NPC) were enrolled from December 2015 to June 2017. Patients with a history of other tumors were excluded. Consent forms in writing were obtained from all subjects. Clinical data were collected for gender, age, diagnosis, pathology type, TNM stage (staging for patients with relapse or distant metastasis referred to TNM stage at NPC initial diagnosis), and plasma EBV DNA level. All patients completed an evaluation before treatment including fiberoptic nasopharyngoscopy, nasopharyngeal and neck magnetic resonance imaging (MRI), chest computed tomography (CT), abdominal ultrasonography and a single photon emission computed tomography (SPECT) whole-body bone scan. Staging of NPC was performed according to the American Joint Committee on Cancer (AJCC) 2010. Abdomen MRI was performed when abdominal ultrasonography suggested the presence of liver metastasis. Twelve NPC patients were subjected to the first-line chemotherapy based on gemcitabine plus cisplatin. After 2 to 4 cycles of chemotherapy, clinical response evaluation was performed according to the Response Evaluation Criteria in Solid Tumors 1.1. Peripheral venous blood (6.0 ml) from each subject was collected into customized tubes containing Acid Citrate Dextrose (ACD)-anticoagulant (Becton Dickinson, Franklin Lakes, NJ, USA) after discarding the first 2.0 ml blood to avoid potential pollutant with skin epithelial cells. All samples were stored at room temperature until been processed within 24 h after collection [19].

Experiment was performed similarly to that published studies and according to the kit instruction (Cytelligen, San Diego, CA, USA) [15, 18]. Briefly, 6.0 ml peripheral venous blood was centrifuged at 800×g for 7 min at room temperature, then, removed supernatants above red blood cells to deplete serum. The left components were thoroughly mixed and overlaid on 3 ml hCTC Separation Matrix. Solutions were centrifuged at 450×g for 6 min at room temperature and white buffy above red blood cells was removed to another centrifuge tube. The collected white buffy was incubated with 150 μl immunomagnetic particles conjugated to anti-leukocytes monoclonal antibodies including anti-CD45 at room temperature for 20 min with gentle shaking. The solution was overlaid on 3.0 ml of hCTC separation matrix, then, centrifuged at 450×g for 5 min at room temperature. Supernatants above magnetic beads were collected and were subsequently subjected to magnetic separation using a magnetic stand (Cytelligen) to remove leukocytes. The bead-free solution was washed with 1 × CRC solution (Cytelligen) and then spun down twice at 700×g for 3 min, 650×g for 3 min at room temperature, separately. Sedimented cells were washed once with 1 × CRC solution (Cytelligen) and centrifuged at 1050×g for 3 min, followed by discarding supernatant. Cell pellets were incubated with 200 μl Antibody Preparation Solution-1, 1 μl Alexa Fluor 594-conjugated monoclonal anti-CD45 and 1 μl Alexa Fluor 488-conjugated anti-EpCAM (Cytelligen) at 30 °C for 20 min in the dark. Sedimented cells were washed once and subsequently centrifuged at 950×g for 3 min at room temperature. The sedimented cells then were thoroughly mixed with cell fixative and applied onto the coated CTC slides (Cytelligen). The slides underwent drying process at 32 °C for 10 h for subsequent iFISH analysis. The slides were soaked in the mixed solution including FR1 and FR2 solution (Cytelligen) at 37 °C for 10 min. Later, the slides followed by 1 × FR3 soaking at 27 °C for 2 min and dehydrated in 100% ethanol for 2 min. Centromere Probe 8 (CEP8) SpectrumOrange (Vysis, Abbott Laboratories, Abbott Park, IL, USA) was denatured at 76 °C for 10 min and hybridized at 37 °C for 4 h. Finally, the slides were soaked in 1 × FR3 solution (Cytelligen) for 2 min and washed once, following stained with 4′,6-diamidino-2-phenylindole (DAPI, Vector laboratories, CA, USA) and subsequently subjected to fluorescence microscope (Carl Zeiss, Jena, Germany).

From December 2015 to June 2017, a total of 50 patients with confirmed nasopharyngeal carcinoma including twenty-nine newly diagnosed patients without distant metastasis (M0) and twenty-one relapsed or distant metastatic patients after 6 months of initial treatment were enrolled in this study. The median age of all patients was 48 (range from 17 to 66) years. Forty-nine patients were the World Health Organization (WHO) II-type or III-type, and only one patient was the WHO I-type. Among the 29 newly diagnosed (M0) patients, 93.1% (27/29) of patients were classified as stage III/IV. No patient with stage I was enrolled. Clinical characteristics of 50 patients were shown in Table 1.

All patients′ blood samples were subjected to subtraction enrichment (SE), following by EpCAM-iFISH analysis. We were inspired by previous studies in other cancer types. CTCs were identified as nucleated cells (DAPI+) with epithelial markers (EpCAM+) and/or aneuploidy (CEP8+) but without WBC marker (CD45-) [15, 18, 19]. Specifically, CTCs were defined as EpCAM+/CD45-/DAPI+/CEP8 ≥ 2, EpCAM-/CD45-/DAPI+/CEP8 > 2. Nucleated cells with CD45+ was defined as WBC. CTM was defined as a cluster of 2 or more CTCs (Fig. 1).

According to above definition, CTCs were detected in 46 cases (92.0%) among the 50 NPC patients, and CTM were observed in 6 patients (12.0%) in this study. The positive rate of CTCs in newly diagnosed (M0) patients and relapsed or distant metastatic patients was 93.1% (27/29) and 90.5% (19/21), respectively. CTM positive rate was 10.3% (3/29) and 14.3% (3/21) in these two groups of patients separately. No significant association was observed between CTCs positive rate and clinical parameters including gender, age, pathological type, and TNM stage (Table 2).

In the present study, CTCs number per 6.0 ml peripheral blood in 50 patients was 0–45 (mean ± SD, 5.8 ± 9.5). CTCs number was 0–24 CTCs/6.0 ml (mean ± SD, 3.7 ± 4.6 CTCs/6.0 ml) in the 29 newly diagnosed patients without distant metastasis (M0) and 0–45 CTCs/6.0 ml (mean ± SD, 8.6 ± 13.3 CTCs/6.0 ml) in the 21 relapsed or distant metastatic patients. CTCs number in relapsed/distant metastatic patients was more than in newly diagnosed (M0) patients. This difference was no statistically significance (P = 0.117). TNM stage was an important clinical factor for NPC patients. To evaluate the correlation of CTCs number and clinical stage in NPC, we compared the relationship between CTCs number and T-classification, N-classification and TNM stage in this study (Table 3). In the newly diagnosed (M0) patients, CTCs number per 6.0 ml peripheral blood in stage II/III/IV were 0.5 ± 0.7 (range, 0–1), 3.7 ± 3.4 (range, 0–13), and 4.1 ± 5.7 (range, 1–24), respectively. Similarly, CTCs number of relapsed or distant metastatic patients in stage II/III/IV were 2.5 ± 2.1 (range, 1–4), 8.6 ± 13.3 (range, 0–12), and 12.0 ± 16.8 (range, 0–45), respectively. Staging of relapsed/distant metastatic patients referred to TNM stage at NPC initial diagnosis. CTCs number constantly increased with TNM stage rising (from stage II to stage IV) no matter in newly diagnosed (M0) patients and relapsed/distant metastatic patients. Nevertheless, CTCs number was not significantly associated with T-classification or N-classification.

The changes of CTCs number were investigated in the 12 NPC patients who underwent the first-line gemcitabine plus cisplatin chemotherapy, including 7 newly diagnosed (M0) patients and 5 relapsed or distant metastatic patients. Correlation of CTCs number to therapeutic efficacy was shown in Fig. 2a. After 2 to 4 cycles of chemotherapy, the number of CTCs decreased from 8.8 ± 13.1 (range, 1–45) to 4.3 ± 7.2 (range, 0–26), and the difference was statistically significant (P = 0.049). Additional analysis was performed on those 12 cases who were subdivided into 2 groups based on their responses to treatment: partial response (PR, 30% or more decrease in the sum of diameters of target lesions), and progressive disease or stable disease (PD, at least 20% increase in the sum of diameters of target lesions; SD, less 30% decrease and less 20% increase in the sum of diameters of target lesions). CTCs number in PR patients dramatically decreased from 10.8 ± 14.8 (range, 1–45) to 4.7 ± 8.3 (range, 0–26) following treatment, with statistically significance (P = 0.040), as shown in Fig. 2b. Whereas, instead of decrease, CTCs number in PD/SD patients even slightly increased from 2.7 ± 1.5 (range, 1–4) to 3.0 ± 1.7 (range, 2–5) after chemotherapy. It was also noticed that CTCs were disappeared from 2 patients with PR following chemotherapy. Obtained results suggested that alterations of CTCs number were correlation to treatment responses, and monitoring CTCs number could predict therapeutic efficacy.

Based on the in situ karyotyping of SE-iFISH, aneuploidy of chromosome 8 in CTCs of NPC patients prior to treatment was examined. We evaluated the relationship between CTCs karyotyping and disease staging. In this study, diploid, triploid, tetraploid, and multiploid (pentaploid or > 5 copies of chromosome 8) CTCs were observed in the 50 NPC patients. The ratio of CTCs with different ploids of chromosome 8 were 2.0% (diploidy), 72.0% (triploidy), 46.0% (tetraploidy) and 58.0% (multiploidy) in all pre-treatment patients. For 29 newly diagnosed (M0) patients, the ratio of CTCs with different karyotypes were 82.8% (triploidy), 44.8% (tetraploidy) and 48.3% (multiploidy), while no diploid CTCs was found in those patients, as shown in Fig. 3a. Relationship between CTCs karyotyping and disease staging was further analysed. Results indicated that triploidy was the largest comparing with other karyotypes of CTCs in newly diagnosed (M0) patients, and the ratio of triploid CTCs were 50.0, 75.0, and 93.3% in stage II/III/IV of those patients, respectively (Fig. 3b). However, the frequency of CTCs with different karyotypes in 21 relapsed or distant metastatic patients were 4.8% (diploidy), 57.1% (triploidy), 47.6% (tetraploidy) and 66.7% (multiploidy), shown in Fig. 3c. Noticeably, the percentage of multiploid CTCs was the most in patients with relapse or distant metastasis. Furthermore, the ratio of multiploid CTCs was increasing in later stage patients with relapse or distant metastasis (Fig. 3d). We also studied the relationship between CTCs number with different karyotypes and disease staging in NPC, shown in Table 4. Triploid CTCs number was the most in newly diagnosed (M0) patients, while multiploid CTCs was the most in patients with relapse or distant metastasis. No significant difference was revealed between CTCs number with different karyotypes and TNM stage in NPC.

We monitored the changes of CTCs karyotyping of the 12 NPC patients post to chemotherapy based on gemcitabine plus cisplatin. For those patients prior to treatment, the ratio of CTCs with different chromosome 8 copy numbers were 8.3% (diploidy), 83.3% (triploidy), 66.7% (tetraploidy) and 41.7% (multiploidy), shown in Fig. 4a. Significantly decreased frequency was observed in tetraploid CTCs (66.7 to 33.3%), whereas decreased frequency of both triploidy (83.3 to 75.0%) and multiploidy (41.7 to 33.3%) was lower than tetraploidy. In addition, the frequency of diploid CTCs remained the same (8.3% versus 8.3%) after treatment. Additional analysis was performed on those 12 patients according to their responses to treatment, shown in Fig. 4b and c. The ratio of triploid CTCs decreased from 88.9% (before treatment) to 66.7% (post treatment) in PR patients, however, increased from 66.7.0% (before treatment) to 100% (post treatment) in PD/SD patients. For tetraploid CTCs, the ratio decreased from 66.7% (before treatment) to 33.3% (post treatment) in PR patients, and the same result was observed in PD/SD patients, from 66.7% (before treatment) to 33.3% (post treatment). In addition, the ratio of multiploid CTCs in PR patients was also decreased from 44.4% (before treatment) to 33.3% (post treatment), but still remained the same (33.3% versus 33.3%) in PD/SD patients after chemotherapy. In summary, following gemcitabine plus cisplatin chemotherapy, the ratio of tetraploid CTCs decreased obviously in all of 12 patients. Nevertheless, the ratio of triploid CTCs just decreased slightly in PR patients, but increased significantly in PD/SD patients. And the percentage of multiploid CTCs decreased in PR patients, but remained the same in PD/SD patients. Noticeably, those patients with PD/SD were relapsed/distant metastatic NPC patients. Results suggested that unlike sensitivity to chemotherapy for tetraploid CTCs, triploid and multiploid CTCs might be susceptible to drug resistance. Triploid and multiploid CTCs may correlate to chemo-resistance in relapsed/distant metastatic NPC patients.

NPC has been proven as an EBV-associated carcinoma, and most previous studies have established that plasma EBV DNA level is a significant prediction marker of distant metastasis in NPC [20, 21]. We evaluated the relationship between CTCs number and plasma EBV DNA level. Among the 67 tested samples including 50 pre-treatment and 17 post-treatment patients, the positive rate of plasma EBV DNA was 80.6% (54/67), and CTCs positive rate was 88.1% (59/67). Slight positive correlation was found between CTCs count and plasma EBV DNA level. The Pearson linear correlation coefficient (R) between them was 0.326, and the association was statistically significant (P = 0.007), shown in Fig. 5a. For 50 NPC patients prior to treatment, the positive rate of plasma EBV DNA was 86.0% (43/50), CTCs positive rate was 92.0% (46/50). Additional analysis about the 50 pre-treatment samples of NPC revealed that positive correlation was also found between CTCs number and plasma EBV DNA level prior to treatment, with statistically significant (R = 0.345, P = 0.014), shown in Fig. 5b. Interestingly, seven pre-treatment patients with negative EBV DNA level were patients with detectable CTCs, and 5 cases of those were confirmed as patients with relapse or distant metastasis. Results indicated that CTCs number in peripheral venous blood had a positive correlation with plasma EBV DNA level of NPC patients, and detecting CTCs by use of SE-iFISH was more sensitive than plasma EBV DNA level test.