Research review
Quantitative RT-PCR Detection of Colorectal Tumor Cells in Peripheral Blood—A Systematic Review

https://doi.org/10.1016/j.jss.2008.02.012Get rights and content

Background

Twenty percent to 40% of patients with node-negative colorectal cancer die of metastatic disease. Detection of cancer cell dissemination has been proposed as a tool to select patients at highest risk for recurrence. In this review, we summarize the evidence for detection with quantitative reverse transcriptase-polymerase chain reaction (RT-PCR) assays of circulating tumor cells (CTCs) in peripheral blood of colorectal cancer patients.

Materials and methods

Pubmed and Embase were systematically searched for all English publications relevant to circulating cancer cells, peripheral blood, quantitative RT-PCR (q-RT-PCR), and colorectal cancer. Cross-references and the “related articles” function were used to broaden the search. Manuscripts reporting on the results of nonquantitative RT-PCR assays were excluded. The study methodology, CTCs detection rates in peripheral blood, and prognostic value were reviewed.

Results

Twelve manuscripts on qRT-PCR were retrieved. Stage dependence was found for detection of CTCs in four of 10 studies. From univariate analysis performed for disease-free survival and overall survival in 4 of 12 studies, there was evidence (P < 0.05) for an effect of the detection of CTCs with qRT-PCR. None of the included trials identified detection of CTCs in peripheral blood as an independent predictor of survival.

Conclusion

Quantification of CTCs in peripheral blood holds promise in predicting stage and outcome in colorectal cancer patients. At present, evidence from the literature is too scarce to integrate quantitative RT-PCR assays to detect CTCs into the management of colorectal cancer.

Introduction

Metastatic lesions and not the primary tumors are the leading cause of death in patients with carcinomas [1]. After curative surgery, 20% to 40% of patients with so-called localized colorectal cancer die of metastasis [2, 3]. During this metastatic process, cancerous cells detach from the primary tumor and migrate to secondary organs. Over almost a century, circulating tumor cells (CTCs) have been a focus of research in the measurement of this phenomenon.

Initially, detection of CTC in blood of cancer patients was achieved by cytology, immunocytochemistry (ICC), and flow cytometry based techniques. Cytology allowed detection of CTCs in the blood in 25% to 67% of examined patients [4, 5, 6, 7, 8]. The combination of cytology and ICC permitted to detect CTCs in four of 42 patients undergoing resection for colorectal cancer [9]. These three techniques have high specificity but limited sensitivity [10].

Reverse transcriptase-polymerase chain reaction (RT-PCR), a modification of classic PCR, was designed to detect mRNA. RT-PCR identification of mRNA coding for CEA, CK20, k-ras mutations, CD44 splice variants, and other proteins is capable of identifying cultured colorectal carcinoma cells in normal blood with a sensitivity of 1 in 107 leukocytes (1–10 mL of blood) [11, 12, 13, 14, 15, 16]. This represents a level of sensitivity three orders of magnitude greater than ICC [17]. Multimarker RT-PCR has further enhanced the assay sensitivity. The use of more than one marker gene eliminates some of the problems associated with single-marker detection techniques, such as tumor cell heterogeneity, clonal selection during tumor metastasis, and variable expression of individual genes within tumor cells [18]. This enhanced sensitivity, however is at the cost of lowering specificity if detection of just one of these markers is considered a positive result.

The detection of CTCs in perioperative venous blood with nonquantitative RT-PCR assays was found to have prognostic significance in some studies [19, 20, 21, 22, 23, 24, 25, 26]. However, the majority of studies failed to show a predictive value. Inconsistent findings can be explained by a lack of statistical power and inadequate length of follow-up. Other explanations for inconclusive results are high background expression of the selected markers resulting in false-positive results and the genetic heterogeneity of tumor cells. Moreover, traditional RT-PCR assays are at most a semiquantitative measurement of cancer cell load in the peripheral blood. Quantitative RT-PCR (qRT-PCR) is another modification of the PCR-principle and eliminates at least some of the problems associated with traditional RT-PCR. These assays, whether TaqMan or SyBGreen based platforms, allow for quantification of cancer cell load in peripheral blood and determination of cutoff values indicating mRNA expression levels of clinical relevance in cancer patients compared with healthy subjects [27]. Using fluorescent dyes that preferentially bind to double-stranded DNA, quantification of mRNA expression of a specific gene in a sample can be accomplished when it is compared with a control sample [28]. An additional advantage of qRT-PCR is the possibility to take into account variations in RNA and/or cDNA quality by quantifying housekeeping genes and subsequent normalization of marker concentration to that of the housekeeping gene [29].

In this manuscript we review the present evidence for quantification of CTCs in peripheral blood of colorectal cancer patients with qRT-PCR.

Section snippets

Materials and Methods

Pubmed and Embase were systematically searched for English articles published between January 1993 and January 2008. The following search terms were used for Pubmed: “neoplasm circulating cells” [MeSH Terms] AND “colorectal neoplasm” [MeSH Terms] AND “peripheral blood” [All Fields] AND (“reverse transcriptase-polymerase chain reaction” [MeSH Terms] OR “quantitative pcr” [All Fields] OR “real-time pcr” [All Fields]) NOT “leukemia” [All Fields]. Search terms for Embase were: ((‘real time

Clinicopathological Characteristics

One of 12 original articles in this review was a multicenter study (Table 1). All studies included adenocarcinomas of the colon and rectum.

Timing and Origin of Blood Sample

In 8 studies, peripheral blood samples were taken prior to treatment (Table 2). In 11 studies, venous blood samples were used to quantify CTCs. A single study used blood from the radial artery [32]. Intra-operative mesenteric venous blood samples were taken in two studies. In three studies, the timing of blood samples was not reported. Volume of blood

Detection of Circulating Tumor Cells

In recent years, many studies have addressed the feasibility and clinical impact of detecting minimal residual disease in lymph nodes, bone marrow, peritoneal cavity, liver, and blood of patients with colorectal cancer. The peripheral blood in particular has practical advantages over other compartments since it can be reached by a routine venipuncture. Although conflicting results have been obtained, several studies suggest the potential of CTC detection in the peripheral blood for cancer

Conclusions

Although studies in the past have shown conflicting results, more and more evidence points toward a clinical relevance for quantification of solid tumor cells circulating in peripheral blood of colorectal cancer patients. Further research including standardization of study methodology is needed before circulating tumor cell quantification can be implemented into the routine clinical setting.

Acknowledgments

GS acknowledges support by a doctoral grant from the Institute for the Promotion of Innovation through Science and Technology in Flanders (IWT Vlaanderen).

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