Originally published as JCO Early Release 10.1200/JCO.2005.04.5948 on June 12 2006

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Predictive and Prognostic Value of Peripheral Blood Cytokeratin-19 mRNA-Positive Cells Detected by Real-Time Polymerase Chain Reaction in Node-Negative Breast Cancer Patients

Nikos Xenidis, Maria Perraki, Maria Kafousi, Stella Apostolaki, Irene Bolonaki, Aliki Stathopoulou, Kostas Kalbakis, Nikos Androulakis, Charalambos Kouroussis, Thanos Pallis, Charalambos Christophylakis, Katerina Argyraki, Evi S. Lianidou, Stathis Stathopoulos, Vassilis Georgoulias, Dimitris Mavroudis

From the Departments of Medical Oncology Pathology, University General Hospital of Heraklion; Laboratory of Tumor Cell Biology, School of Medicine, University of Crete, Heraklion, Crete; and the Laboratory of Analytical Chemistry, Faculty of Chemistry, University of Athens, Athens, Greece.

Address reprint requests to Vassilis Georgoulias, MD, PhD, Department of Medical Oncology, University General Hospital of Heraklion, PO Box 1352, 711 10 Heraklion, Crete, Greece; e-mail: georgsec{at}med.uoc.gr

	ABSTRACT

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Purpose: To evaluate the predictive and prognostic value of peripheral blood cytokeratin-19 (CK-19) mRNA-positive cells in axillary lymph node–negative breast cancer patients.

Patients and Methods: Peripheral blood was obtained from 167 node-negative breast cancer patients before the initiation of any systemic adjuvant therapy, and was analyzed for the presence of CK-19 mRNA-positive cells using a real time polymerase chain reaction assay. The association with known prognostic factors and the effect of CK-19 mRNA-positive cells on patients' prognosis was investigated.

Results: CK-19 mRNA-positive cells were detected in the blood of 36 (21.6%) of the 167 patients. There was no correlation between the detection of CK-19 mRNA-positive cells in the peripheral blood and the various known pathologic and clinical prognostic factors; only overexpression of HER2 receptor (score 2+/3+) on the primary tumor was associated with a higher incidence of CK-19 mRNA-positive cell detection (P = .033). Multivariate analysis revealed that detection of peripheral blood CK-19 mRNA-positive cells was associated with early clinical relapse (P < .00001) and disease-related death (P = .008).

Conclusion: Detection of peripheral-blood CK-19 mRNA-positive cells is an independent predictive and prognostic factor for reduced disease-free interval and overall survival, respectively, in node-negative breast cancer patients.

	INTRODUCTION

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In the recent years, breast cancer is increasingly diagnosed at an early stage because of the massive use of screening mammography programs. Indeed, 56.2% of all breast cancer cases diagnosed in 1995 were stage 0 or 1 compared with 42.5% in 1985.^1,2 Adjuvant chemotherapy and hormone treatments have improved disease-free interval and overall survival.³ Nevertheless, nearly 30% of patients with node-negative (N0) breast cancer will present distant recurrence and will die as a result of their disseminated disease,³ whereas 40% of the patients with involved axillary lymph nodes will survive for 10 years without clinical recurrence.⁴

The concept of early tumor cell dissemination in patients with early-stage breast cancer was born about 100 years ago from Paget's "seed and soil" theory.⁵ According to this hypothesis, metastasis is a result of rare cells migrating from the primary tumor through the lymphatic or hematogenous route. In breast cancer, tumor cell dissemination to the regional lymph nodes occurs through the lymphatic vasculature⁶ but may also occur directly through the hematogenous route; it seems that different biologic mechanisms are required for the lymphatic or hematogenous dissemination of tumor cells.^7,8

The detection of occult tumor cells in the bone marrow or peripheral blood of patients with early-stage breast cancer has been shown to be an independent predictive and prognostic factor for early disease recurrence and decreased overall survival.^9-13 Recently, Braun et al⁴ reported that the immunohistochemical detection of bone marrow–disseminated tumor cells (DTCs) in 4,703 patients with operable breast cancer was an independent prognostic parameter for early relapse and death during the first 5 years of follow-up.¹⁴

So far, the clinical relevance of occult tumor cells in breast cancer patients was studied in bone marrow aspirates using immunohistochemical assays.^9-11,13 However, bone marrow is not an easy source for monitoring occult tumor cells. Using a nested reverse-transcriptase (RT-) polymerase chain reaction (PCR) assay, we have previously shown in a cohort of 148 patients with stage I and II operable breast cancer that peripheral blood cytokeratin-19 (CK-19) mRNA-positive cells (circulating tumor cells [CTCs]) before the initiation of adjuvant treatment was an independent predictive and prognostic factor for early relapse and overall survival, respectively.¹²

Real-time PCR is an automated, rapid, versatile, and cost-effective technology capable of providing accurate and quantitative information about gene expression.¹⁵ This method has been used for the detection of CTCs in the peripheral blood and lymph nodes of metastatic breast cancer patients through either differential¹⁶ or multigene¹⁷ expression.

In the present study, we investigated the presence of CK-19 mRNA-positive CTCs using a highly sensitive and specific real-time PCR assay¹⁸ in a cohort of N0 breast cancer patients before the initiation of any adjuvant treatment. The data demonstrated that detection of CK-19 mRNA-positive CTCs in these patients was an independent predictive and prognostic factor for early recurrence and decreased overall survival, respectively.

	PATIENTS AND METHODS

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Patients and Clinical Samples
Peripheral blood (10 mL in EDTA) was obtained from 167 patients with N0 breast cancer on the day of initiation of adjuvant treatment (usually 3 to 4 weeks after primary surgery). All blood samples were obtained at the middle of vein puncture after the first 5 mL of blood were discarded. This precaution was undertaken to avoid contamination of the blood sample with epithelial cells from the skin during sample collection.

Before primary surgery, a complete baseline diagnostic evaluation for distant metastases was performed, including chest x-rays, mammography, ultrasound of the liver, and whole-body bone scan. Further imaging studies (computed tomography scans or magnetic resonance imaging) were performed if clinically indicated. No patient included in the present analysis showed any evidence of distant metastasis. Most of the patients received adjuvant chemotherapy in the context of research protocols of the Hellenic Oncology Research Group (HORG) especially designed for patients with high-or low-risk N0 breast cancer. Adjuvant chemotherapy consisted of either FEC (fluorouracil 700 mg/m² day 1 + epirubicin 75 mg/m² day 1 + cyclophosphamide 700 mg/m² day 1 every 3 weeks for six cycles) or T/EC (epirubicin 75 mg/m² day 1 + cyclophosphamide 700 mg/m² day 1 every 3 weeks for four cycles followed by docetaxel 100 mg/m² day 1 every 3 weeks for four additional cycles) as well as classical CMF (cyclophosphamide 100 mg/m² orally days 1-14; methotrexate 40 mg/m² days 1 and 8; fluorouracil 600 mg/m² days 1 and 8; every 4 weeks for six cycles). Tamoxifen (20 mg/d) for 5 years was administered to all patients with estrogen-or/and progesterone-positive tumors. Patients were followed with clinical examinations and laboratory and imaging studies every 3 months for the first 2 years, every 6 months for the next 3 years, and yearly thereafter. The study has been approved by the ethics and scientific committees of our institution, and all patients gave their informed consent in order to participate in the study.

CK-19 Immunohistochemistry of Axillary Lymph Nodes
Paraffin-embedded level I and II axillary lymph nodes from patients without axillary lymph node involvement in hematoxylin and eosin staining were further evaluated by immunostaining using an anti–CK-19 (anti-CK-19) mouse antihuman monoclonal antibody (DAKO-CK19, Code#M888; DAKO A/S, Glostrup, Denmark). Anti-CK-19 moA was used at a dilution 1/50 (vol/vol) for 60-minute, after unmasking by heating for 20 minutes, in citrate buffer, pH 6, in a microwave oven at 500 W. The immunostaining method applied was a polymer method based on the DAKO EnVision System, Alkaline Phosphatase, Universal (Code#K1396) with Fast Red substrate-chromogen (Roche Molecular Biochemistry, Manheim, Germany), according to the manufacturer's instructions. Counterstaining was done with hematoxylin and eosin, and coverslipping with an aqueous-based mounting medium (Glycerol, Cook#C-0563, Carpinteria, CA). Known positive and negative controls were always used.

RNA Extraction and Real-Time PCR Assay for CK-19 mRNA-Positive CTCs
Peripheral blood mononuclear cells were obtained by gradient density centrifugation using Ficoll-Hypaque (density 1,077 xg/mol; Sigma-Aldrich, St Louis, MO) at 1,200 g for 30 minutes at 4°C. The interface cells were removed, washed twice with 50 mL of sterile phosphate-buffered saline at 1,200 g for 10 minutes, pelleted, and stored at –80°C until used. Total RNA isolation was performed by using Trizol LS reagent (Gibco, Life Sciences, BRL, Grand Island, NY), according to the manufacturer's instructions. All RNA preparations and handling steps took place in a laminar flow hood, under RNAse-free conditions. The isolated RNA was dissolved in diethylpyrocarbonate-treated water and stored at –80°C until used. RNA concentration was determined by absorbance reading at 260 nm with the Hitachi UV-VIS (U-2000) spectophotometer (Tokyo, Japan). RNA integrity was tested by PCR amplification of the ß-actin housekeeping gene. As positive and negative controls, RNA samples were prepared from the MCF-7 and ARH-77 breast cancer and leukemic cell lines, respectively.

Reverse transcription of RNA was carried out using the Thermoscript RT-PCR system (Invitrogen, Paisley, United Kingdom). cDNA was synthesized according to the manufacturer's instructions. The real-time RT-PCR assay for the detection of CK-19 mRNA-positive cells has already been described.¹⁸ In brief, 2 µl of cDNA were placed into a 18 µl reaction volume containing 1 µl of the sense primer CK-19 for (3 mmol/L), 1µl of the antisense primer CK-19-do (3 mmol/L), 2.4 µl of the LightCycler Fast Start DNA Master Hybridization Probes reagent (10x concentration), 1 µl of the probe CK-19-FL (3 mmol/L), 1 µl of the probe CK-19-LC (3 mmol/L). The primers used have been previously described.¹⁸ PCR reaction was initiated with a 10-minute denaturation at 95°C and terminated with a 30-second cooling step at 40°C. The cycling protocol consisted of a denaturation step at 95°C, annealing at 60°C for 10 seconds, and extension at 72°C and repeated 50 times. Fluorescence detection was performed at the end of each annealing step for less than 1 second. Real-time PCR for the housekeeping gene GAPDH was performed in all of the clinical samples to evaluate the quality of the cDNAs used in the study. The lower detection limit of the assay was set at more than 0.6 MCF-7 cell equivalents/5 µg RNA.¹⁸ The within-run curve values (CVs) for MCF-7 cells as determined by the calibration curve ranged from 7.5% to 9.3%, while the corresponding crossing point (Cp) values from 0.9% to 1.5%; similarly the between-run CVs ranged from 10.7% to 16.0%, whereas the corresponding Cp values ranged from 2.2% to 3.2%.²³ It has been previously shown that this method was highly specific because only two (2.2%) out of 89 blood samples obtained from female blood donors were found to be positive.¹⁸

Statistical Analysis
The main tools of analysis were logistic regression^19,20 and the Cox proportional hazards model²¹ for outcomes related to point events and time variables, respectively. To select those factors with an independent significant influence on outcomes, both analyses were carried out in a stepwise (unconditional backward) fashion.^20,21 Before the application of these methods, univariate analyses were performed for a preliminary exploration of marked associations. Univariate analyses included contingency tables, t or Mann-Whitney U tests, log-rank tests, and simple Cox regression analyses.^21-24

	RESULTS

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Patient Characteristics and Detection of CTCs
Patient characteristics are shown in Table 1. The patients' median age was 49 years (range, 30 to 80 years), 58.1% were premenopausal or perimenopausal (within 12 months from the last menstrual cycle), and 77.8% had a lumpectomy; the primary tumor size was 2.0 cm or smaller in 37.6% of the patients, and 57.2% of the patients had estrogen receptor–negative tumors. A median number of 14 (range, five to 42) axillary level I and II lymph nodes were removed. Immunostaining of lymph node sections with the anti–CK-19 monoclonal antibody could not reveal positive cells, confirming that the study population was indeed N0. All patients received adjuvant chemotherapy (FEC, n = 75; T/EC, n = 51; CMF, n = 39; other miscellaneous regimens, n = 2). Peripheral blood CK-19 mRNA-positive CTCs could be detected by real-time PCR in 36 patients (21.6%) before the initiation of any systemic adjuvant treatment. The median number of CK-19 mRNA-positive cells was 3.4 MCF-7 cell equivalents/5 µg RNA (range, 0.6 to 1,105 equivalent). Detection of CK-19 mRNA-positive CTCs was significantly associated with HER2/neu-positivity (score 2+/3+; P = .033) but not with other patients' or tumors' clinicopathologic characteristics (Table 1).

View larger version (17K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 1. Disease-free interval in cytokeratin-19 (CK-19) mRNA-positive and CK-19 mRNA-negative patients.

View larger version (16K): [in this window] [in a new window] [PowerPoint Slide for Teaching]   Fig 2. Overall survival of cytokeratin-19 (CK-19) mRNA-positive and CK-19 mRNA-negative patients.

	DISCUSSION

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These findings are in agreement with previous studies supporting the prognostic value of occult micrometastatic cells in patients with early-stage (N0 and N1) breast cancer.^10-14,25,26 However, in most of these studies, occult tumor cells were identified in the bone marrow aspirates using an immunohistochemical assay and their clinical relevance was determined in both N0 and node-positive (N1) tumors. Braun et al¹¹ reported that the incidence of both clinical recurrence and disease-related deaths in N0 patients who did not receive systemic adjuvant therapy was higher in the group of patients with detectable DTCs compared with those without detectable DTCs. It is interesting to note that patients with N0 tumors and detectable DTCs had a practically similar median overall survival with that of patients with N1 tumors who did not have detectable DTCs.¹¹ A recent meta-analysis study further confirmed the above prognostic value of detectable CK-19-positive DTCs; indeed, in 1,036 patients with small (T1) and N0 tumors, both the DFI and the overall survival were significantly worse in patients with detectable DTCs compared with those without detectable DTCs.¹⁴ Conversely, Gebauer et al,²⁶ evaluating 194 patients with N0 tumors, could not demonstrate any difference in the incidence of distant recurrences and the rate of cancer-related deaths between the patients with or without detectable DTCs. In addition, Wiedswang et al¹³ reported that among the N0 patients, systemic relapse as well as overall survival did not differ whether or not DTCs were detected; these findings were in overt contrast to those observed in the group of patients with N1 tumors. The reasons for the discrepancies concerning the prognostic and predictive value of DTCs in N0 breast cancer patients are not obvious; we cannot exclude that methodologic reasons that may influence the detection limits of the used assay may account for this phenomenon.

Some of these problems may be eliminated by the development of standardized assays that are sensitive enough to detect a low load of occult tumor cells, thus facilitating the evaluation of the clinical significance of occult tumor cells in N0 breast cancer patients. The high sensitivity and reproducibility of the used real-time PCR assay (0.6 MCF-7 cell equivalents/5 µg RNA) could perhaps explain the highly significant association observed between the detection of CTCs and the risk for clinical recurrence and disease-related death in the present study. Wiedswang et al¹³ used a negatively-selected bone marrow cell population in order to increase the sensitivity of his DTCs detection assay; however, their methodology could not reveal significant differences in patients with N0 tumors. Nevertheless, the superiority of the real-time PCR assay over other assays for the detection of DTCs or/and CTCs should be demonstrated in a prospective comparative study.

Most of the patients enrolled in the studies evaluating the clinical relevance of DTCs mentioned herein^11,13,26 had not received adjuvant chemotherapy. Conversely, patients included in the current study received adjuvant chemotherapy and those with estrogen receptor–or/and progesterone receptor–positive tumors also received tamoxifen. It is interesting to note that the association of the increased risk of relapse as well as of disease-related death and the detection of CTCs before the initiation of any adjuvant treatment was independent of the administered chemotherapy regimen. This observation further supports the hypothesis that detection of either DTCs or CTCs represented a specific biologic characteristic of the tumor, which could be independent of the possibility of some chemotherapy regimens to eliminate bone marrow²⁷ or peripheral blood²⁸ occult tumor cells. However, the administration of CMF was also an independent predictive factor for early relapse when it was compared with the administration of the more intense T/EC regimen. It could be argued that T/EC eliminates CTCs in a significantly higher proportion of patients compared with CMF, thus resulting in a lower incidence of clinical recurrences. These findings should be interpreted with caution until their confirmation with a prospective and well designed study of longitudinal detection of CTCs during the different phases of systemic adjuvant treatment.

Our findings were obtained using RNA extracted from peripheral blood mononuclear cells; therefore, blood could be a valuable source for monitoring CTCs. This opens the way to further investigate important questions such as whether the detection of CTCs should be performed in all patients at the time of primary diagnosis to identify high-risk patients or whether CTCs detection at diagnosis should modify the adjuvant therapeutic strategy, or, finally, whether the detection of CTCs during the administration of adjuvant treatment would allow the development of secondary adjuvant therapeutic strategies. Indeed, several studies have shown the expression of various markers on occult tumor cells, such as uPA/uPAR complex, c-erbB2/neu, and p53, which could serve as targets for tailored treatments.^25-31 We have recently demonstrated that trastuzumab, a humanized monoclonal antibody against the HER2/neu molecule, could eliminate chemotherapy-resistant CTCs and DTCs in 20 of 30 patients with breast cancer.³² Therefore, the study and evaluation of the clinical relevance of CTCs opens the possibility of early and tailored treatment interventions for patients with persisting occult tumor cells after systemic adjuvant treatment.

	Authors' Disclosures of Potential Conflicts of Interest

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The authors indicated no potential conflicts of interest.

	Author Contributions

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION Authors' Disclosures of... Author Contributions Glossary REFERENCES

 

Conception and design: Vassilis Georgoulias Provision of study materials or patients: Irene Bolonaki, Evi S. Lianidou, Stathis Stathopoulos, Vassilis Georgoulias Collection and assembly of data: Nikos Xenidis, Maria Kafousi, Stella Apostolaki, Irene Bolonaki, Aliki Stathopoulou, Kostas Kalbakis, Nikos Androulakis, Charalambos Kouroussis, Thanos Pallis, Charalambos Christophylakis, Katerina Argyraki Data analysis and interpretation: Maria Perraki, Irene Bolonaki, Evi S. Lianidou, Efstathios Stathopoulos, Vassilis Georgoulias, Dimitris Mavroudis Manuscript writing: Irene Bolonaki, Stathis Stathopoulos, Vassilis Georgoulias, Dimitris Mavroudis Final approval of manuscript: Nikos Xenidis, Maria Perraki, Maria Kafousi, Stella Apostolaki, Irene Bolonaki, Aliki Stathopoulou, Kostas Kalbakis, Nikos Androulakis, Charalambos Kouroussis, Thanos Pallis, Charalambos Christophylakis, Katerina Argyraki, Evi S. Lianidou, Stathis Stathopoulos, Vassilis Georgoulias, Dimitris Mavroudis

 

	Glossary

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CK-19 (cytokeratin-19):

CK-19 belongs to the intermediate filaments, which create a cytoskeleton in almost all cells. CK-19 is normally not expressed in the hematopoietic cells, although it is commonly expressed in epithelial cells such as mammary cells, either normal or neoplastic.

CT scan (computed tomography scan):

A series of pictures created by a computer linked to an x-ray machine taken of the inside of the body from different angles.

CTC (circulating tumor cell):

Demonstration of isolated tumor cell circulation/ dissemination in the peripheral blood.

DTC (disseminated tumor cell):

Demonstration of isolated tumor cells disseminated in the bone marrow.

MRI (magnetic resonance imaging):

A procedure in which radio waves and a powerful magnet linked to a computerare used to create detailed pictures of areas inside the body. These pictures can show the difference between normal and diseased tissue.

PBMC (peripheral blood mononuclear cell):

A single-nucleus cell found circulating in the bloodstream (normally includes lymphocytes and monocytes).

RT-PCR (reverse-transcriptase polymerase chain reaction):

PCR is a method that allows logarithmic amplification of short DNA sequences within a longer, double-stranded DNA molecule. Gene expression can be measured after extraction of total RNA and preparation of cDNA by a reverse-transcription step. Thus, RT-PCR enables the detection of PCR products on a real-time basis, making it a sensitive technique for quantitating changes in gene expression.

	NOTES

 
published online ahead of print at www.jco.org on June 12, 2006.

Supported in part by research grants from the Cretan Association of Biomedical Research (CABR). A.S and A.P were recipients of a CABR research fellowship.

Authors' disclosures of potential conflicts of interest and author contributions are found at the end of this article.

Terms in blue are defined in the glossary, found at the end of this article and online at www.jco.org.

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Submitted October 14, 2005; accepted April 5, 2006.

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