Molecular detection of cancer cells in bone marrow and peripheral blood of patients with operable breast cancer. Comparison of CK19, MUC1 and CEA using RT-PCR

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Abstract

We have compared three different RT-PCR procedures to measure cytokeratin 19 (CK19), carcinoembryonic antigen (CEA) and mucin MUC1 gene expression in order to determine their diagnostic value in detecting tumour cells in bone marrow aspirates of patients with operable breast cancer. In an experimental model, the best sensitivity was observed for CK19 and MUC1 RT-PCR assays, although only the CEA and CK19 assays showed good specificity. The study of 42 patients showed that a ‘CK19 positive/CEA positive’ RT-PCR assay in bone marrow correlated positively with a positive axillary lymph node status (N0 versus N1–3, P<0.05). Both assays were also positive in 17% of node negative patients. RT-PCR assays were more sensitive in bone marrow than in peripheral blood. Our results suggest that CK19 and CEA RT-PCR assays are powerful methods for detecting disseminated breast cancer cells. A larger study with long-term follow-up is required in order to clarify their clinical usefullness.

Introduction

Accurate staging of patients diagnosed with breast cancer is important to determine the extent of disease and to plan appropriate therapies. In the last decade there have been remarkable changes in breast cancer treatment. Breast cancer surgery has become more conservative, and an increased number of patients are receiving systemic therapy. The decision to treat breast cancer patients with either chemotherapy or hormonal therapy relies on the tumour size, the nodal status and other prognostic factors. The major problems for prediction of patient's relapse include the heterogeneity of the tumour and the tendency of the disease to spread early. Although 90% of the patients are apparently free of metastases at the time of primary surgery, almost 50% of them will relapse 5 years later [1]. The skeleton represents the most frequent site of metastases caused by breast cancer. These occur in more than 80% of the patients diagnosed with metastatic breast cancer and the skeleton is frequently the first metastatic site to be detected [2]. Therefore, early examination of bone marrow might be helpful in the diagnosis of occult spread of disease.

Using conventional histological methods, disseminated tumour cells are found in only a few cases. The addition of immunohistological methods using monoclonal antibodies (MAbs) directed against surface antigens or cytokeratins has led to improvements in the ability to detect breast cancer cells in bone marrow aspirates and biopsy samples [3]. More recently, the use of polymerase chain amplification to identify cell-specific messenger RNA (RT-PCR) has become a highly sensitive tool in the detection of minimal involvement of the blood and bone marrow in a variety of malignancies, including melanoma [4], neuroblastoma [5], prostate cancer [6] and breast cancer [7]. The choice of the messenger RNA (mRNA) target for amplification is determined by the specific characteristics of the malignant cells, but it should not be expressed in bone-marrow cells, lymph node cells or peripheral blood cells. Noguchi and colleagues [8] reported that the expression of the gene coding for the mucin MUC1 is suitable for the detection of disseminated breast cancer cells in lymph nodes. Gerhard and colleagues [9] demonstrated that the carcinoembryonic antigen (CEA) is a good target gene for detecting abdominal cancer or breast cancer cells in the bone marrow. Several workers agree that the detection of cytokeratin 19 (CK19) mRNA by RT-PCR is a useful test in order to find a few disseminated tumour cells in the bone marrow of patients with breast cancer 10, 11. We have shown that RT-PCR for CK19 is a reliable procedure that detects up to one human breast cancer T47D cell per 106 peripheral blood mononuclear (PBMN) cells, with higher sensitivity (and the same high specificity) as two different CK19 immunostaining methods [12].

To evaluate if measurement of these three different mRNAs by different mRNA by RT-PCR assays (MUC1, CEA and CK19) have a similar diagnostic value in detecting the presence of disseminated breast cancer cells we compared these procedures in reconstitution experiments that mimic clinical samples, as well as in bone marrow and peripheral blood samples obtained from breast cancer patients before the operation.

Section snippets

Cell samples

The human mammary carcinoma cell line T47D was cultured in Dulbecco's modified Eagles medium (DMEM) supplemented with 10% bovine fetal serum (FBS), 2 mM L-glutamine and 1 mM pyruvate. We harvested the T47D cells grown in monolayer by washing the dishes once with phosphate-buffered saline (PBS) and then incubating the cells with PBS containing 0.53 mM EDTA and 0.05% trypsin (Gibco, Grand Island, USA) for 5 min at 37°C. After washing in PBS, the cells were passed into syringes (25 G 5/8 needle)

Sensitivity and specificity of each RNA-based diagnosis

To establish the limits of sensitivity and specificity of the three RT-PCR assays for tumour cell detection, T47D cells were seeded into different tubes containing PBMN cells. A total of 10 million cells were present in each sample at tumour cell — haematopoietic cell ratios ranging from 1:103 to 1:107, thus mimicking the clinical setting for detection of mammary cells in patient's peripheral blood or bone marrow. An additional 20 non-seeded PBMN specimens obtained from different healthy

Discussion

Detection of small numbers of breast cancer cells in patient blood and bone marrow has become increasingly important. The ability to rule out the presence of micrometastatic disease at any stage of the clinical management protocol, whether before, during, or after therapy, would provide a useful monitoring and diagnostic tool. In the past few years it has been reported that the RT-PCR is a method with high sensitivity and could be applicable to the diagnosis of minimal residual disease in

Acknowledgements

This work was supported by grants from Comisión Honoraria de Lucha Contra el Cáncer (Uruguay). We thank Ivana Facini for her helpful contribution.

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