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

doi:10.1016/S0304-3835(00)00630-3

Cancer Letters

Volume 162, Issue 1, 10 January 2001, Pages 65-73

https://doi.org/10.1016/S0304-3835(00)00630-3 Get rights and content

Abstract

We prospectively analyzed the circulating tumor burden in colorectal cancer patients using semi-quantitative reverse transcription-polymerase chain reaction (RT-PCR) for carcinoembryonic antigen (CEA) and cytokeratin 19 (CK19 ). We distinguished the mRNA levels in peripheral blood between 33 patients and 26 healthy controls with reference to SK-BR-3 cell line. We found CEA-mRNA in 88% of patients and 92% of controls, and CK19 mRNA in 64% of patients and 19% of controls. Our CK19 mRNA assay was sufficiently sensitive to detect one SK-BR-3 cell among 10⁶ normal blood cells. The upper limit of CK19 mRNA among controls was exceeded by 14 patients, and 12 patients (86%) developed systemic metastases/recurrence. Significantly elevated CK19 mRNA levels appeared to originate from circulating malignant cells (P<0.0001). Of relevance, the CK19 mRNA level increased with advancing Dukes' stage and correlated directly with the serum CEA level (P=0.016). CK19 mRNA quantification may prove valuable for cancer staging and disease monitoring.

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.

Section snippets

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.

References (35)

F. Lindemann et al.
Prognostic significance of micrometastatic tumour cells in bone marrow of colorectal cancer patients
Lancet
(1992)
P. Anker et al.
K-ras mutations are found in DNA extracted from the plasma of patients with colorectal cancer
Gastroenterology
(1997)
I.H.N. Wong et al.
Aberrant p15 promoter methylation in adult and childhood acute leukemias of nearly all morphologic subtypes: potential prognostic implications
Blood
(2000)
N. Frickhofen et al.
Rearranged Ig heavy chain DNA is detectable in cell-free blood samples of patients with B-cell neoplasia
Blood
(1997)
F. Arakawa et al.
Characterization of a cDNA clone encoding a new species of the non-specific cross-reacting antigen (NCA), a member of the CEA gene family
Biochem. Biophys. Res. Commun.
(1990)
P. Chomczynski et al.
Single-step method of RNA isolation by acid guanidinium thiocyanate- phenol-chloroform extraction
Anal. Biochem.
(1987)
B. Greenway
Hepatic metastases from colorectal cancer: resection or not
Br. J. Surg.
(1988)
I.H.N. Wong et al.
Quantitative analysis of circulating tumor cells in peripheral blood of osteosarcoma patients using osteoblast-specific mRNA markers: a pilot study
Clin. Cancer Res.
(2000)
I.H.N. Wong et al.
Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients
Cancer Res.
(1999)
I.H.N. Wong et al.
Tumor-derived epigenetic changes in the plasma and serum of liver cancer patients: implications for cancer detection and monitoring
Ann. N Y Acad. Sci.
(2000)

T. Yamada et al.

Detection of K-ras gene mutations in plasma DNA of patients with pancreatic adenocarcinoma: correlation with clinicopathological features

Clin. Cancer Res.

(1998)

Y. Fujiwara et al.

Plasma DNA microsatellites as tumor-specific markers and indicators of tumor progression in melanoma patients

Cancer Res.

(1999)

Y.H. Datta et al.

Sensitive detection of occult breast cancer by the reverse transcriptase polymerase chain reaction

J. Clin. Oncol.

(1994)

M. Gerhard et al.

Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction

J. Clin. Oncol.

(1994)

A.M. Dingemans et al.

Detection of cytokeratin-19 transcripts by reverse transcriptase-polymerase chain reaction in lung cancer cell lines and blood of lung cancer patients

Lab. Invest.

(1997)

S. Noguchi et al.

Detection of breast cancer micrometastases in axillary lymph nodes by means of reverse transcriptase-polymerase chain reaction. Comparison between MUC1 mRNA and keratin 19 mRNA amplification

Am. J. Pathol.

(1996)

M. Mori et al.

Molecular detection of circulating solid carcinoma cells in the peripheral blood: the concept of early systemic disease

Int. J. Cancer

(1996)

Cited by (47)

Regional Liver Therapy Using Oncolytic Virus to Target Hepatic Colorectal Metastases
2010, Seminars in Oncology
Citation Excerpt :
In our group's NV1020 trial, all patients experienced partial response and CEA reduction following viral and chemotherapy, and the patient with the greatest radiographic tumor reduction in response to one dose of virus alone (39%) also had the greatest reduction of CEA level (75%).17,21 While CEA levels vary greatly between patients, relative change in CEA in a single patient has been correlated with tumor recurrence and tumor burden.81–84 Accordingly, CEA could serve as a marker for oHSV efficacy during treatment, perhaps guiding future dosing strategies in individual patients.
The mortality of colorectal carcinoma often results from the progression of metastatic disease, which is predominantly hepatic. Although recent advances in surgical, locoregional, and systemic therapies have yielded modest improvements in survival, treatment of these aggressive lesions is limited to palliation for the vast majority of patients. Oncolytic viral therapy represents a promising novel therapeutic modality that has achieved tumor regression in several preclinical and clinical models. Evidence further suggests that locoregional viral administration may improve viral efficacy while minimizing toxicity. This study will review the theories behind hepatic arterial infusion of oncolytic virus, as well as herpes viral design, preclinical data, and clinical progress in regional liver therapy using oncolytic virus to treat hepatic colorectal carcinoma metastases.
Why the need for qPCR publication guidelines?-The case for MIQE
2010, Methods
The polymerase chain reaction (PCR) has matured from a labour- and time-intensive, low throughput qualitative gel-based technique to an easily automated, rapid, high throughput quantitative technology. Real-time quantitative PCR (qPCR) has become the benchmark technology for the detection and quantification of nucleic acids in a research, diagnostic, forensic and biotechnology setting. However, ill-assorted pre-assay conditions, poor assay design and inappropriate data analysis methodologies have resulted in the recurrent publication of data that are at best inconsistent and at worst irrelevant and even misleading. Furthermore, there is a lamentable lack of transparency of reporting, with the “Materials and Methods” sections of many publications, especially those with high impact factors, not fit for the purpose of evaluating the quality of any reported qPCR data. This poses a challenge to the integrity of the scientific literature, with serious consequences not just for basic research, but potentially calamitous implications for drug development and disease monitoring. These issues are being addressed by a set of guidelines that propose a minimum standard for the provision of information for qPCR experiments (“MIQE”). MIQE aims to restructure to-day’s free-for-all qPCR methods into a more consistent format that will encourage detailed auditing of experimental detail, data analysis and reporting principles. General implementation of these guidelines is an important requisite for the maturing of qPCR into a robust, accurate and reliable nucleic acid quantification technology.
A herpes oncolytic virus can be delivered via the vasculature to produce biologic changes in human colorectal cancer
2009, Molecular Therapy
Citation Excerpt :
CEA is the standard tumor marker used to assess the presence of colorectal cancer.21 Although absolute levels vary greatly from patient to patient, the relative change in level in a single patient has been shown to correlate with tumor recurrence22,23,24 and tumor burden.25 Changes in CEA have also been shown to be indicative of tumor response to therapy that can be seen before radiologic changes.
Genetically engineered herpes simplex viruses (HSVs) can selectively infect and replicate in cancer cells, and are candidates for use as oncolytic therapy. This long-term report of a phase I trial examines vascular administration of HSV as therapy for cancer. Twelve subjects with metastatic colorectal cancer within the liver failing first-line chemotherapy were treated in four cohorts with a single dose (3 × 10⁶ to 1 × 10⁸ particles) of NV1020, a multimutated, replication-competent HSV. After hepatic arterial administration, subjects were observed for 4 weeks before starting intra-arterial chemotherapy. All patients exhibited progression of disease before HSV injection. During observation, levels of the tumor marker carcinoembryonic antigen (CEA) decreased (median % drop = 24%; range 13–74%; P < 0.02). One of three individuals at the 10⁸ level showed a 39% radiologic decrease in tumor size by cross-section and 75% by volume. HSV infection was documented from liver tumor biopsies. After beginning regional chemotherapy, all patients demonstrated a further decrease in CEA (median 96%; range 50–98%; P < 0.008) and a radiologic partial response. Median survival for this group was 25 months. During follow-up, no signs of virus reactivation were found. Multimutated HSV can be delivered safely into the human bloodstream to produce selective infection of tumor tissues and biologic effects.
Molecular Detection of Circulating Tumor Cells
2009, Cell and Tissue Based Molecular Pathology
Molecular Detection of Circulating Tumor Cells
2008, Cell and Tissue Based Molecular Pathology: A Volume in the Foundations in Diagnostic Pathology Series
Multipoint quantification of multimarker genes in peripheral blood and micrometastasis characteristic in peri-operative esophageal cancer patients
2008, Cancer Letters
Citation Excerpt :
This work further proposed a method to reveal the metastatic characteristic of tumor cells with several mRNA expressions and their combination in peripheral blood by multimarker genes and multipoint quantification. Although different marker genes, such as CK19 [6], CEA [7], CK20 [8] and survivin mRNA [9], have been singly used to investigate the occult metastasis in peripheral blood (PB) based on RT-PCR technology, no marker gene has been found to be consistently and specifically expressed in all of the primary tumors for a particular malignancy [10]. Thus multiple marker genes in PB have been applied recently to improve the detection sensitivity of breast cancer [10], melanoma [11] and lung cancer cells [12–14], which increases the positivity detection rate by 32% [10] or to 72% [12].
This work proposed a method to assess the occult micrometastasis characteristic in peri-operative peripheral blood (PB) based on multipoint quantification of multimarker genes by real-time semiquantitative RT-PCR. The expression levels of SCC, CK19, CK20, CEA and survivin mRNA in PB samples collected before surgery (B − 1), immediately after surgery (B0) and at the third day post-operatively (B + 3) from 36 squamous esophageal cancer (EC) patients were detected. SCC and CK19 mRNA showed low positivity detection rates, while the rate for CEA and survivin mRNA panel was 58.3%, 83.3% and 72.3% at B − 1, B0 and B + 3, respectively. Opposite to the significant increase and slow decrease of CEA cells at stages of B − 1 to B0 and B0 to B + 3, respectively, survivin cells decreased significantly and increased quickly at the two stages. The follow-up with a period of 1.2 years showed that the patients with the B + 3/B0 ratios of ⩾0.3 for CEA cells or/and ⩾10 for survivin cells exhibited significantly high possibility of developed metastasis, and the sensitivity to predict developed metastasis increased from 54.5% of CEA mRNA alone to 72.7% of CEA and survivin mRNA panel. These results suggested that CEA and survivin gene panel improved the sensitivity to predict recurrence and differentiate the micrometastatic process.

View all citing articles on Scopus

View full text

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

Abstract

Introduction

Section snippets

Patients and control subjects

Detection of CEA and CK19 mRNAs in PBNCs from healthy subjects

Discussion

Acknowledgements

Lancet

Gastroenterology

Blood

Blood

Biochem. Biophys. Res. Commun.

Anal. Biochem.

Hepatic metastases from colorectal cancer: resection or not

Br. J. Surg.

Quantitative analysis of circulating tumor cells in peripheral blood of osteosarcoma patients using osteoblast-specific mRNA markers: a pilot study

Clin. Cancer Res.

Detection of aberrant p16 methylation in the plasma and serum of liver cancer patients

Cancer Res.

Tumor-derived epigenetic changes in the plasma and serum of liver cancer patients: implications for cancer detection and monitoring

Ann. N Y Acad. Sci.

Detection of K-ras gene mutations in plasma DNA of patients with pancreatic adenocarcinoma: correlation with clinicopathological features

Clin. Cancer Res.

Plasma DNA microsatellites as tumor-specific markers and indicators of tumor progression in melanoma patients

Cancer Res.

Sensitive detection of occult breast cancer by the reverse transcriptase polymerase chain reaction

J. Clin. Oncol.

Specific detection of carcinoembryonic antigen-expressing tumor cells in bone marrow aspirates by polymerase chain reaction

J. Clin. Oncol.

Detection of cytokeratin-19 transcripts by reverse transcriptase-polymerase chain reaction in lung cancer cell lines and blood of lung cancer patients

Lab. Invest.

Detection of breast cancer micrometastases in axillary lymph nodes by means of reverse transcriptase-polymerase chain reaction. Comparison between MUC1 mRNA and keratin 19 mRNA amplification

Am. J. Pathol.

Molecular detection of circulating solid carcinoma cells in the peripheral blood: the concept of early systemic disease

Int. J. Cancer