Quantitative relationship of the circulating tumor burden assessed by reverse transcription-polymerase chain reaction for cytokeratin 19 mRNA in peripheral blood of colorectal cancer patients with Dukes' stage, serum carcinoembryonic antigen level and tumor progression
Introduction
Mortality and morbidity among patients with colorectal cancer (CRC) is mainly caused by tumor dissemination via the bloodstream or lymphatic circulation. Unfortunately, micrometastases could be missed by histopathologic examination of specimens used for tumor staging. Nearly 40% of CRC patients without bone marrow micrometastasis develop clinical metastasis/recurrence [1], [2]. It has been demonstrated that the presence of epithelial tumor cells in bone marrow of CRC patients may have prognostic significance [2]. For early diagnosis of metastasis/recurrence, the highly sensitive PCR can be clinically useful to detect the circulating tumor load in cancer patients [3], [4], [5], [6], [7], [8], [9], [10].
Application of more than one mRNA markers may enhance the sensitivity and specificity of reverse transcription-PCR (RT-PCR) in diagnosing micrometastasis. Immunocytochemistry using anti-cytokeratin antibodies has been applied for detecting micrometastasis. Cytokeratin 19 (CK19) mRNA of epithelial origin appears to be an appropriate molecular marker for CRC [11]. To monitor metastasis/recurrence, levels of serum carcinoembryonic antigen (CEA, an epithelial cell surface glycoprotein) are generally measured for CRC patients. CEA mRNA that is presumably expressed in epithelial cells could be another potential marker [12]. Although immunocytochemistry data are basically concordant with RT-PCR results [13], RT-PCR has been proven much more sensitive for detecting micrometastasis or circulating tumor cells [11], [12], [13], [14]. However, the clinical relevance of CEA mRNA remains controversial. The CEA mRNA detection rate in blood has been associated with disease stage [15]. On the other hand, no direct correlation was found between the presence of CEA mRNA in blood and clinicopathologic features [16], [17].
The presence of CEA and CK19 mRNAs in blood from healthy/non-cancer controls appears to be attributed to illegitimate transcription in normal hematopoietic cells [13], [16], [18], [19]. Also, CK19 is expressed in fibroblasts, endothelial cells and keratinocytes [20]. The problem of illegitimate transcription or skin contamination during venepuncture may possibly limit the specificity of RT-PCR. To minimize this problem, we have applied a quantitative approach for the precise assessment of the circulating tumor burden and hence the risk for metastasis/recurrence [4], [21], [22].
In this prospective study, we assessed the diagnostic value of semi-quantitative RT-PCR for CEA and CK19 mRNAs in CRC patients. As CEA- and CK19 -homologous genes have been identified [23], [24], [25], [26], we applied specifically designed PCR primers [11], [12], [25]. To differentiate CEA/CK19 mRNA from the homologous genes, DNase I treatment was conducted prior to RT-PCR to remove pseudogenes and contaminating genomic DNA. Also, we applied Southern blot analysis to verify the CEA/CK19 identity and enhance the assay specificity and sensitivity. Using a quantitative approach, we distinguished the mRNA levels in peripheral blood between CRC patients and healthy subjects with reference to an epithelial tumor-derived cell line, SK-BR-3. To investigate the clinicopathologic relevance of RT-PCR data, we correlated the mRNA levels in patients blood with serum CEA levels, tumor stages, and clinical metastases/recurrence.
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Patients and control subjects
With informed consent and approval from the Ethics Committee of the Chinese University of Hong Kong, we prospectively collected peripheral blood samples from 33 CRC patients (female to male ratio=12:21; median age=64 years, range: 24–80 years). According to Dukes' staging, initial tumor stage was categorized. After surgical resection, all the CRC patients were treated with chemotherapy and/or radiotherapy according to standardized protocols. All the patients were followed up regularly at 3–6
Detection of CEA and CK19 mRNAs in PBNCs from healthy subjects
CEA mRNA signals were obtained in PBNCs from 92% (24/26) of healthy subjects (Fig. 1). Also, CEA mRNA was detected in 88% (29/33) of CRC patients. In contrast, CK19 mRNA signals were obtained in PBNCs from only five of 26 (19%) healthy subjects (Fig. 1). The CK19 mRNA detection rate among the controls was much lower than that for CEA mRNA. Therefore, CK19 mRNA appeared to be a more specific marker for CRC. We determined the upper limit of CK19 mRNA for the controls (mean+3 SD), and set this as
Discussion
We found CK19 mRNA superior to CEA mRNA as a molecular marker in a significant proportion of CRC patients. In contrast to the absence of CEA mRNA in normal/non-tumorous marrow or blood as reported previously [12], [15], we and others have detected CEA mRNA in blood from healthy or non-cancer subjects [16], [19]. This discrepancy may be related to different techniques (nested PCR and Southern blot analysis) and RT-PCR conditions applied. As CEA-homologous genes are expressed in granulocytes and
Acknowledgements
This work was supported by grants from the Strategic Grants Council and the Direct Grants Council from the Chinese University of Hong Kong.
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