Abstract
Background: The study was designed to detect disseminated tumor cells (DTCs) in postoperative peripheral blood of patients with oral squamous cell carcinoma (OSCC) and to determine their relevance as prognostic markers by cytokeratin (CK) expression analysis. Materials and Methods: Forty samples of peripheral blood mononuclear cells (PBMCs) isolated 4 weeks after surgery were screened for occurrence of four CK mRNA transcripts by real-time quantitative RT qPCR. Detection of mRNA expression was compared with clinicopathological parameters and disease-free survival (DFS). Results: CK 17 and CK 19 could not be detected in any samples. CK 18 and CK 20 were detectable in 1 (2.5%) and in 14 (35.0%), respectively. The detection of CK 20 was not significantly associated with lymph node status, clinical stage, or differentiation grade, but was significantly higher in patients with T3 and T4 OSCC (p=0.04). DFS was not associated with tumor size, clinical stage, or differentiation grade. But poor DFS was significantly associated with the occurrence of lymph node metastasis (p=0.01) and detection of CK 20 (p=0.01). Conclusion: DTCs in PBMCs of postoperative patients with OSCC could only be detected by determination of CK 20 mRNA. Detection of CK 20 mRNA in peripheral blood seems to be of relevance for prognosis in OSCC.
- Oral squamous cell carcinoma
- cytokeratin
- disseminated tumor cells
- peripheral blood
- SYBR green real-time quantitative reverse transcriptase-polymerase chain reaction
Despite diagnostic and therapeutic advances by the introduction of multimodal therapy, the 5-year survival rate of patients with oral squamous cell carcinoma (OSCC) remains poor, often due to recurrence or metastatic disease (1-5). The failure of any reduction of morbidity most likely results from early dissemination of tumor cells into bone marrow or peripheral blood, which is usually missed by conventional staging procedures at the time of surgery and leads to recurrence and metastatic disease. Since adjuvant therapy aims to eradicate occult disseminated tumor cells (DTCs) before recurrence and metastatic disease becomes clinically evident, there is an urgent demand to develop strategies for the detection of DTCs to enable clinicians to identify patients who would benefit from early systemic treatment.
In the past, cytokeratins (CKs) have been used as molecular markers for the detection of DTCs in patients suffering from solid tumors because they are constitutively expressed in epithelial cells and often overexpressed in tumor cells, but their expression cannot normally be found in hematopoietic cell compartments such as bone marrow and peripheral blood (6-9). CK expression has been examined in the primary tissues, bone marrow and peripheral blood of patients suffering from solid tumors, including head and neck carcinoma. The expression rates are associated with prognosis (10-14).
Recently, CK 17 was identified as a diagnostic marker for primary OSCC (15) and as a specific immunohistochemical marker in SCC of the larynx, in breast carcinoma and in cervical carcinoma (16-18). Using immunohisotochemistry, detection of CK 18 overexpression in tumor tissues of the oral cavity was associated with a poor clinical outcome (19). The detection of CK 19 mRNA in tissues by real-time quantitative reverse transcriptase-polymerase chain reaction (RT qPCR) may be a prognostic marker for OSCC (20). In addition, CK 19 and 20 were extensively investigated for detection of DTCs in peripheral blood and bone marrow in patients suffering from breast carcinoma, colorectal carcinoma and pancreatic carcinoma (10, 21-24). Moreover, CK 20 mRNA expression in primary tissues of OSCC was associated with the occurrence of metastases of neck lymph nodes (15).
However, there has been only one study on the detection of CK 20 mRNA in peripheral blood of patients suffering from OSCC and in a quite small number of samples so that the postulated association between CK 20 mRNA and tumor recurrence could not be evaluated statistically (25). Hence, there have been no extensive studies on detection of DTCs in OSCC by determination of CK gene expression in order to assess the patient's prognosis.
The aim of this study was to determine the occurrence of mRNA transcripts of CK 17, CK 18, CK 19 and CK 20 by real-time RT qPCR in peripheral blood mononuclear cells (PBMCs) of a greater number of patients suffering from OSCC to ascertain whether these CKs would be suitable markers both for detection of DTCs and prognosis.
Materials and Methods
Patients and sample collection. The study comprised PBMC samples from 40 patients suffering from OSCC of different TNM classification and stages and from 5 otherwise healthy volunteers acting as calibrator in real-time RT qPCR. The study was approved by the local Ethical Committee and the patients gave their informed consent.
Peripheral blood for the study was obtained approximately 4 weeks after surgery. After discarding the first 5 ml of blood to avoid contamination with epidermal cells, a total of 20 ml of blood was collected with heparin. The blood was diluted with 20 ml phosphate-buffered saline (PBS). Blood constituents were separated by density centrifugation with Ficoll-Paque Plus (Amersham Biosciences, Germany) and PBMCs harvested from the interphase were washed twice in PBS. PBMCs were then shock frozen in liquid nitrogen and stored at -80°C until real-time RT qPCR analyses.
All patients from whom the samples were obtained were examined clinically and staged according to the TNM classification and to the UICC (26). Differentiation grades were classified according to the WHO (27). Clinical information including age, gender, TNM classification, clinical stage and differentiation grade, and disease-free endpoints were reviewed from the records of the department of Oral and Maxillofacial Surgery, University of Erlangen-Nuremberg. Tumor recurrence was excluded by chest X-ray, bone scan and abdominal and pelvic ultrasound. Patient survival was evaluated within a period of a maximum of 156 months (range, 20-156 months; median, 43.5 months).
Detection of CK 17, CK 18, CK 19 and CK 20 mRNA by real-time reverse transcriptase polymerase chain reaction. Total RNA from PBMCs was extracted using commercially available TRIzol® (Invitrogen, Karlsruhe, Germany) according to the manufacturer's protocol. After DNase treatment for 1 hour at 37°C, the isolated total RNA was cleaned up with RNeasy Kit (QIAGEN, Hilden, Germany). cDNAs from total RNA were synthesized using with the High Capacity cDNA Archive Kit (Cat. 4322171; Applied Biosystems, CA, USA) according to the manufacturer's protocol. Real-time RT qPCR analyses were performed using QuantiTect Primer Assay (200) [Hs KRT17 1 SG QuantiTect Primer Assay (200) (Cat. QT00001680) for CK 17, Hs KRT18 1 SG QuantiTect Primer Assay (200) (Cat. QT01003562) for CK 18, Hs KRT19 1 SG QuantiTect Primer Assay (200) (Cat. QT00081137) for CK 19, and Hs KRT20 1 SG QuantiTect Primer Assay (200) (Cat. QT00014784) for CK 20 QIAGEN]. For normalization, GAPDH was used [Hs GAPDH 1 SG QuantiTect Primer Assay (200) (Cat. QT00079247); QIAGEN]. The relative quantification (RQ) of mRNA was performed with the ABI Prism 7300 Sequence Detection System (Applied Biosystems, CA, USA). The QuantiTect TM SYBR® green PCR kit (Cat. 204143; QIAGEN) was used for PCR amplification. In total, 40 ng of cDNA were used for each PCR reaction in a total volume of 25 μl. Each PCR run included a 15 min activation time at 95°C. The three-step cycle was run as follows: denaturing at 94°C for 15 s, annealing at 55°C for 30 s and extension at 72°C for 34 s. Formation of undesired by-products during PCR that contribute to fluorescence was assessed by melting curve analysis after PCR. CK 17, CK 18, CK 19 and CK 20 mRNA quantities were analysed in duplicate, normalised against GAPDH as an internal control gene and expressed in relation to mRNA isolated from PBMCs of healthy volunteer as a calibrator. Relative gene expression was determined using the ΔΔCt method (28). RNA isolated from squamous cell carcinoma cell line of the head and neck SCC 9 was used as a positive control.
Statistics. The statistical analyses were performed using SPSS version 14.0 software (SPSS, Chicago, IL, USA). The mean value of duplicate CK mRNA RQs was defined to be positive if it was higher than 2.0-fold. The Chi-square test or the log-rank test was used to assess any correlations between the overexpression of CK mRNA and clinicopathological parameters. Kaplan-Meier analysis was used to evaluate the follow-up data of the 40 patients. P-values of less than 0.05 were considered to be significant.
Results
Forty patients suffering from OSCC were included in the study. Patient characteristics are given in Table I.
Detection of CK 17, CK 18, CK 19, and CK 20 mRNA in peripheral blood. Using our selection criteria, CK 17 and CK 19 mRNA could not be detected, since all values of RQ were lower than 2.0-fold. CK 18 mRNA was detected in only 1 out of 40 samples (2.5%). On the other hand, CK 20 mRNA was detected in 14 out of 40 (35.0%) (Table I). The results from 10 representative patients show that CK 20 mRNA was satisfactorily detected by the real-time RT qPCR (Figure 1). Focusing on CK 20 mRNA detection in PBMCs, the detection rate was not significantly associated with gender, age, state of lymph node, clinical stage, or differentiation grade, but was significantly higher in patients suffering from T3 and T4 OSCC (p=0.04) (Table II).
Follow-up and univariate analysis of prognostic factors. The patients were followed up for a maximum of 156 months (range, 20-156 months; median, 43.5 months). Survival analysis of patients shows that gender, age, tumor size, clinical stage and differentiation grade were not associated with survival rate (Table III). However, both the occurrence of metastases of neck lymph nodes and detection of CK 20 mRNA in PBMCs were significantly associated with poor disease-free survival (p=0.01) (Figures 2, 3).
Discussion
It is generally accepted that DTCs occur at low frequencies and cannot easily be detected in peripheral blood circulation, even when advanced metastatic disease is present in patients (29-31). DTCs are found in the central venous blood and bone marrow of head and neck carcinoma patients at a much lower frequency compared to those in patients with other carcinomas (11, 14, 32). These incidences are presumed to be the result of sampling errors, intermittent tumor-cell shedding, or effective immune surveillance, or to reflect stochastic effects at the lower limit of sensitivity of the assay.
Real-time RT-PCR is a sensitive and specific method for analysing gene expression patterns in tissues and in body fluids. CKs are constitutively expressed in epithelial cells and highly overexpressed in tumor specimens. In an otherwise healthy person, they are absent in hematopoietic cell compartments such as the bone marrow and the peripheral blood (6-9). Therefore it should be possible to detect DTCs deriving from epithelial tissues based on levels of CK mRNA expressions. In this study, a sensitive real-time RT qPCR was carried out, offering the possibility to detect 1 to 10 tumor cells mixed with 1 million normal mononuclear cells (33, 34). Each CK mRNA RQ was compared to that of healthy volunteers to determine the specific overexpression in PBMCs of patients suffering from OSCC. False-positive results due to contamination of PBMCs samples with dermal cells at needling were avoided by internal rinsing of the catheter and removing the first 5 ml blood aspirate. Thus, occurrence of normal epithelial cells in peripheral circulation was ruled out. The detection of CK mRNA in isolated PBMCs indicated the occurrence of tumor cells disseminated into the peripheral circulation from the primary site with high probability.
Due to its high overexpression in primary tissues compared to that in normal mucosa, CK 17 mRNA seems to be one of the most suitable diagnostic markers for OSCC (15-18). CK 18 overexpression in the oral cavity was also found to be an independent prognostic marker and indicated a lower overall and progression-free survival (19). In addition CK 19 and CK 20 have been extensively examined for detection of DTCs in breast, colorectal and pancreatic carcinoma (10, 21-24). In contrast, the results of this study show that only CK 20 mRNA could be detected at a high rate in peripheral blood of patients suffering from OSCC, whereas CK18 and CK17/19 could only be detected in 2.5% and in none of the blood samples, respectively. Hence, in contrast to detection of tumor cells in primary tissues, these results confirm that CK 20 could be the most suitable molecular marker for detection of DTCs deriving from primary tissues of OSCC in peripheral circulation by real-time RT qPCR. These results are supported by studies using CK 20 for detection of DTCs in peripherial blood in patients suffering from colorectal and pancreatic carcinoma (10, 12, 21, 22, 24). Additionally, CK 20 expression was also shown in 11 out of 12 patients suffering from OSCC (25). Indeed an association was shown between hematogenous CK 20 mRNA detection and recurrence in patients suffering from OSCC, but the number of patients was too small for a statistical evaluation. Moreover, a diagnostic and prognostic relevance of CK 20 mRNA detection in lymph nodes by RT qPCR was demonstrated in patients suffering from OSCC and colorectal cancer (35-37). In the present study, a significant increase in survival rate was found for patients who were examined negatively for CK 20 mRNA expression in PBMCs. This confirms the prognostic relevance of CK 20 detection in hematopoietic cell compartments postoperatively. Combining the prognostic relevance with its clinical utility as a marker, we believe that detection of CK 20 mRNA could be used to develop strategies for screening of DTCs in peripheral blood of postoperative patients. Occurrence of CK 20-positive DTCs could serve to predict recurrence and metastatic disease in patients as early as possible and to improve the patient's prognosis. Further studies on a greater number of patients are needed to evaluate the results.
In the previous study, overexpression of CK 20 mRNA in the primary tissues of OSCC was significantly associated with the occurrence of metastases of neck lymph nodes (15). In this study, the detection rate of CK 20 mRNA in the peripheral blood was significantly associated with tumor size, if T1 and T2, and T3 and T4 were grouped together. Hence the expression rate of CK 20 in the primary site may increase following tumor growth. Furthermore, it cannot be ruled out that in the majority of cases, tumor cells starting to express the CK 20 gene may disseminate from the primary site and develop recurrence or metastatic disease in other organs (11, 13, 21, 22, 36).
In conclusion, the presence of DTCs in peripheral blood of patients suffering from OSCC could only be detected by determination of CK 20 mRNA expression. The expression of CK 20 was significantly higher in T3 and T4 OSCC. Poor DFS was significantly associated with the occurrence of lymph node metastasis and detection of CK 20. Thus, out of all examined CKs only the detection of CK 20 mRNA in peripheral blood seems to be a suitable additional marker for detection of DTCs and to be of relevance to a patient's prognosis.
Acknowledgements
This study was supported by a grant of the Johannes und Frieda Marohn-Stiftung Erlangen (TOY/2006).
Footnotes
- Received May 13, 2008.
- Revision received August 20, 2008.
- Accepted November 6, 2008.
- Copyright© 2009 International Institute of Anticancer Research (Dr. John G. Delinassios), All rights reserved