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Detection of Circulating Mammary Carcinoma Cells in the Peripheral Blood of Breast Cancer Patients Via a Nested Reverse Transcriptase Polymerase Chain Reaction Assay for Mammaglobin mRNA

From the First Internal Department, Elisabethinen Hospital, Linz, Austria.

Address reprint requests to O. Zach, PhD, Elisabethinen Hospital, First Internal Department, Fadingerstr 1, A-4010 Linz, Austria; email dieter.lutz{at}elisabethinen.or.at

	ABSTRACT

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
PURPOSE: According to current medical research, mammaglobin (hMAM) is expressed exclusively in the mammary glands of adult women and in mammary tumor cell lines. Therefore, we examined hMAM expression as a marker for the detection of carcinoma cells in the peripheral blood of patients with breast cancer (BC).

PATIENTS AND METHODS: Blood samples obtained from 114 BC patients at the various stages of their disease and from 68 individuals without BC were screened for hMAM mRNA by a nested reverse transcriptase polymerase chain reaction (RT-PCR) assay.

RESULTS: The assay exhibited a calculated analytical limit of one tumor cell per 10⁶ to 10⁷ WBCs. None of the samples from peripheral blood of 27 healthy individuals were positive, whereas 29 (25%) of 114 samples from BC patients were positive for hMAM mRNA. hMAM mRNA expression was detected in five (28%) of 18 BC patients at diagnosis, in three (6%) of 53 with no evidence of disease, and in 21 (49%) of 43 with metastatic disease. These results correlate with patients' carcinoembryonic antigen (CEA) plasma level and, to some extent, with estrogen receptor status. Two of 41 samples from patients with malignancies other than BC were also positive.

CONCLUSION: In contrast to healthy volunteers, hMAM transcripts were detected in the peripheral blood of BC patients. The percentage of positivity relates to the clinical stages of disease, CEA plasma level, and estrogen receptor status. Aberrant hMAM expression might occur occasionally in malignancies other than BC. The clinical relevance of hMAM RT-PCR–based tumor cell detection in the peripheral blood of BC patients should be further evaluated in prospective studies.

	INTRODUCTION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
THE IDENTIFICATION of circulating tumor cells in the peripheral blood of breast cancer (BC) patients could potentially become an important prognostic factor for survival, because early dissemination of tumor cells is one of the main causes for disease progression.¹ Because cytologic staining methods have low sensitivity, immunocytologic tests were developed that were able to increase sensitivity but with some degree of false positivity.²

In the early 1990s, several molecular biologic techniques based on reverse transcriptase polymerase chain reaction (RT-PCR) have been tested for their ability to detect minimal residual breast carcinoma cells. These assays used the expression of certain genes, primarily carcinoembryonic antigen (CEA)^3-5 and cytokeratin 19,^6-10 as markers for the presence of tumor cells. Both genes are of low specificity for tumor cells, as transcripts are occasionally detected in the blood, lymph nodes, and bone marrow of healthy volunteers.^11-15 Therefore, mRNA expression of these genes has limited diagnostic value as a marker for the detection of micrometastases by the RT-PCR assay.¹⁵

In 1996, the cDNA for a novel gene termed human mammaglobin (hMAM) was isolated.¹⁶ The amino acid sequence of hMAM exhibits homology to several secreted epithelial proteins of the uteroglobin gene family, but the cellular function of this protein has not been clarified yet. The hMAM gene is localized to chromosome 11q13, a region that is amplified in a subset of breast neoplasia.¹⁷ As far as it is known, the expression of hMAM is restricted to the adult mammary gland and to mammary tumor cell lines, and it is overexpressed in 23% of primary human breast tumors compared with normal breast tissue.¹⁶ Northern blot analysis of 17 cell lines of various origin, including malignant ones, revealed expression of hMAM mRNA only in the subset of BC cell lines (high expression in MDA-MB415 cells and low expression in MDA-MB175, but no expression was detected in MCF7 cells).¹⁶ Additionally, of 16 human tissues (including breast, ovary, uterus, and peripheral-blood leukocytes) tested for hMAM mRNA molecules via a one-step RT-PCR assay, the only positive sample was derived from breast tissue.¹⁶

Because the expression of hMAM was discovered to be mammary-specific, we used it as a marker gene for cells derived from breast tissue and developed a nested RT-PCR assay for the detection of hMAM mRNA in the peripheral blood of BC patients.¹⁸

	PATIENTS AND METHODS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Patients and Cell Line
Blood samples from 27 healthy volunteers, 41 patients with various malignancies other than BC (as controls), and 114 BC patients were collected after informed consent was given. BC patients (median age, 54 years; range, 26 to 79 years) were classified into three defined clinical subgroups: 18 patients were considered to be at diagnosis (Dx; after surgery, without metastasis, and before any further adjuvant treatment), 53 had no evidence of disease (NED; stages I to III or relapsed and after chemotherapy-induced remission), and 43 had metastatic disease (MD; stage IV or relapse of earlier stages). Thirty-four patients with NED had been off treatment for at least 3 months and up to 5 years, 17 patients received chemotherapy, and two patients received hormone treatment when samples were taken, whereas 37 patients with MD received chemotherapy and the remaining six received hormone treatment only. Estrogen receptor (ER) status (tested immunocytologically) and CEA plasma levels (measured via an enzyme immunoassay) were taken from patient records. None of the peripheral-blood samples were tested immunocytologically for other tumor markers. The SKBR5 human mammary carcinoma cell line was a generous gift of W. Scheirer (Novartis, Vienna).

RNA Extraction and cDNA Synthesis
Circulating tumor cells in the peripheral blood of BC patients are very few (if they are present at all) and require sensitive methods for detection. Accordingly, hMAM mRNA molecules may be rare compared with WBC-derived mRNA and might become undetectable in patients with high WBC counts when using fixed quantities of RNA for cDNA synthesis. To overcome this problem, constant volumes of RNA solutions prepared from 5 mL of peripheral blood were used for first-strand cDNA synthesis, independent of the total RNA amount. Therefore, detection of hMAM mRNA is not influenced by varying WBC counts of our patients at the time when blood samples were taken.

Erythrocytes of 2 x 5 mL EDTA blood per patient were lysed as described⁸; nucleated cells were spun down and dissolved in 1 mL of TRIZOL reagent (Life Technologies, Vienna, Austria). RNA was isolated according to the manufacturer's instructions and dissolved in 50µL of diethylpyrocarbonate-treated water. RNA was quantified spectrophotometrically at 260 nm and stored at -20°C. Two microliters of RNA solution (range, 0.2 to 5 µg; mean, 1.1 ± 0.7 µg) was subjected to reverse transcription with 200 units of SUPERSCRIPT II RT (Life Technologies) and 0.5 µg of oligo (dT) primer (Pharmacia-Biotech, Vienna, Austria) in a volume of 20 µL for 50 minutes at 42°C. Probes were stored at 4°C until use.

PCR and Analysis of Products
In all PCR assays, cDNA probes from SKBR5 cells and from healthy volunteers were used as positive and negative controls, respectively. Four microliters of freshly prepared cDNA from each RNA preparation was subjected two times to PCR, resulting in four PCR setups for each assay per patient. PCR was carried out in a volume of 50 µL containing 10-fold–concentrated reaction buffer (Perkin Elmer, Norwalk, CT), 200 µmol/L of each nucleotide (Pharmacia-Biotech), 0.5 µmol/L of each primer, and 1.5 units of AmpliTaq DNA Polymerase (Perkin Elmer). All primers were synthesized at the MWG-Biotech laboratory (Ebersberg, Germany); sequences are listed in Fig 1. The nested primers for hMAM were designed to generate a PCR product spanning the whole translated region of its mRNA. Due to two introns located in this area, amplification of genomic DNA or unspliced mRNA generates a 2.814–base pair (bp) product, whereas mature hMAM mRNA molecules produce a fragment of 201-bp length.¹⁷ The cycle conditions were as follows: 2 minutes at 95°C, 30 cycles of 15 seconds at 95°C, 15 seconds at 62°C, 20 seconds at 72°C, and, lastly, 7 minutes at 72°C. Thermal cycling was performed in a GeneAmp 2400 apparatus (Perkin Elmer). Two microliters of the first amplification product was subjected to a second amplification using the inner primer pairs and the same cycle conditions as described above. The presence of intact RNA and adequate cDNA synthesis was confirmed by a single-round RT-PCR using beta-2 microtubulin–specific primers¹² and cycling conditions as for hMAM. Each sample was subjected to electrophoresis with 1.5% agarose gels stained with ethidium bromide. Samples of each patient were considered to have a positive score if at least one of the four lanes showed a band of the expected size.

View larger version (13K): [in this window] [in a new window]   Fig 1. Primer sequences for the hMAM nested RT-PCR assay were derived from the published sequence with GenBank accession no. U33147.16 The primers were designed to generate a PCR product that spans the whole translated region of hMAM mRNA. Primer pair MG-1, MG-2 was used for the first amplification; MG-3, MG-4 was used for the second.

Sample Preparation for Determination of Analytical Limit
Four hundred cells of the BC cell line SKBR5 were transferred into 10 mL of peripheral blood of healthy volunteers with a WBC count of 5.5 to 10.8 x 10⁶/mL. Five milliliters of the samples were repeatedly diluted with 5 mL of peripheral blood until reaching a calculated concentration of 3 SKBR5 cells in 5 mL of peripheral blood. RNA was prepared from 5 mL of each dilution (as described under RNA Extraction and cDNA Synthesis), and the nested RT-PCR assay was performed.

Statistical Analysis
Two-tailed Fisher's exact probability test was used for the statistical analysis.

	RESULTS

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
Detection of hMAM Transcripts in Peripheral-Blood Samples
An amplified hMAM product of SKBR5 mammary carcinoma cells is shown in Fig 2 (lane 8). When dilutions of SKBR5 cells in peripheral blood of healthy volunteers were tested, a calculated analytical limit for the hMAM assay of one SKBR5 cell in 10⁶ to 10⁷ WBCs was regularly achieved.

View larger version (49K): [in this window] [in a new window]   Fig 2. Ethidium bromide–stained 1.5% agarose gel of amplified beta-2 microtubulin (lanes 1 to 6) and hMAM (lanes 8 to 20) PCR products. Two RNA preparations were made from each patient, and each preparation was subjected to one beta-2 microtubulin and two hMAM RT-PCR setups. Samples were derived from a patient at Dx stage III (lanes 1 to 2 and 9 to 12), a patient with MD stage IV (lanes 3 to 4 and 13 to 16), and a patient at Dx stage I (lanes 5 to 6 and 17 to 20). Lane 7, 100-bp DNA ladder (GIBCO-BRL, Vienna, Austria); lane 8, mammary carcinoma cell line SKBR5. Both patients at stages III and IV were positive for hMAM mRNA, whereas the stage I patient was negative. The length of PCR products was 201 bp for hMAM and 268 bp for beta-2 microtubulin.

RNA samples prepared from peripheral blood of all 27 healthy individuals, including 10 lactating women, were negative for hMAM transcripts (Table 1). Furthermore, we analyzed the peripheral blood of 41 patients with malignancies other than BC (Table 1), and 39 of these samples were negative. The two positive samples were derived from female patients undergoing chemotherapy for thymus carcinoma and mantle-cell lymphoma (stage IV).

Detection of hMAM Transcripts in Peripheral Blood of BC Patients
Amplified products of the nested RT-PCR assay for hMAM of three BC patients are shown in Fig 2. Twenty-nine peripheral-blood samples from 114 BC patients were positive for hMAM (25%). The results in detail were as follows: five (28%) of 18 BC patients at Dx, three (6%) of 53 with NED, and 21 (49%) of 43 with MD had detectable hMAM mRNA in their peripheral blood. The P values for the differences between these clinical subgroups are .02 for Dx/NED, .000001 for NED/MD, and .16 for Dx/MD (Table 2).

ER status was tested for the primary tumors of 99 BC patients. Thirty percent (16 of 54) of ER-positive and 16% (seven of 45) of ER-negative patients were positive for hMAM mRNA in their peripheral blood (P = .15). However, a difference between ER-positive and ER-negative patients is only seen in subgroups of patients at Dx and with MD but not in patients with NED (Table 3).

The CEA plasma level at the time when samples were taken was available for 80 BC patients only. Sixteen percent (10 of 61) of patients with CEA < 3.5 ng/mL were hMAM mRNA–positive, whereas 53% (10 of 19) of BC patients with CEA 3.5 ng/mL were hMAM mRNA–positive (P = .005). hMAM mRNA was detected in all three defined clinical subgroups of BC patients with low CEA plasma levels (Table 3).

	DISCUSSION

TOP ABSTRACT INTRODUCTION PATIENTS AND METHODS RESULTS DISCUSSION REFERENCES

 
The ability to detect malignant spread at its early stage is desirable, because this may have important prognostic and therapeutic implications. Currently tumor metastasis is diagnosed by clinical manifestations and imaging studies together with serum marker assays. These techniques are only useful in later stages of tumor spread because of the need for a critical minimal tumor volume.¹⁹ Sensitive immunocytologic tests for the detection of single tumor cells in the peripheral blood of cancer patients were developed. However, the antibodies used had some degree of false positivity, and this, therefore, limited their prognostic value.²

Tissue-specific gene expression allows the use of RT-PCR in the detection of restricted mRNA species in secondary sites like peripheral blood, which is compatible with metastatic spread.¹⁹ The detection of mRNA for tumor-specific genes (eg, CEA) or epithelium-specific genes (eg, cytokeratin 19)^3-10 as markers for the presence of tumor cells in the peripheral blood of BC patients has been shown to exhibit limited diagnostic value, because occasionally cells that are positive for these markers are detected in the peripheral blood of healthy volunteers.^11-15 Therefore, tissue and/or tumor specific marker genes are needed for the detection of metastases in the peripheral blood of BC patients.

hMAM-expressing cells were detected by RT-PCR amplification in the peripheral blood of BC patients but not in that of healthy individuals, which indicates high specificity of hMAM as a marker gene for cells derived from mammary glands. The incidence of detectable hMAM transcripts in peripheral blood of BC patients is correlated with subgroups (28% Dx, 6% NED, and 49% MD) and, in part, with CEA plasma levels and ER status. Most of the hMAM-positive patients had metastatic disease and/or elevated CEA plasma levels. However, hMAM mRNA was also detected in some of the patients without advanced disease (Dx and NED) or with low CEA plasma levels. It remains to be evaluated in prospective studies whether tumor cells in peripheral blood detected by hMAM expression are of any prognostic value, especially for patients at Dx without advanced disease and for patients with NED.

When SKBR5 mammary carcinoma cells were diluted in peripheral blood of healthy volunteers, the calculated analytical limit of the hMAM nested RT-PCR assay reached one SKBR5 cell in 10⁶ to 10⁷ WBCs; even the hMAM expression level of this cell line is much lower than, for example, that of MDA-MB415 cells (N. Mudie, personal communication). In this context, it must be noted that some mammary tumors show overexpression of hMAM mRNA¹⁶ and that the amount of hMAM mRNA molecules in peripheral-blood samples of BC patients might depend not only on the number of tumor cells but also on the extent of hMAM mRNA expression per single cell. Therefore, the individual detection limit for hMAM mRNA may vary from patient to patient. In addition, it is not yet known whether all BC cells express hMAM at all.

No hMAM mRNA expression was detected in 39 of 41 peripheral blood samples from patients with malignancies other than BC. The remaining two patients with positive hMAM RT-PCR assays had lymphoid malignancies, ie, thymus carcinoma and mantle-cell lymphoma. The t(11;14)(q13;q32) rearrangement that involves the chromosomal region where the hMAM gene is located has been reported in various types of lymphomas and is a characteristic translocation of mantle-cell lymphomas.²⁰ No karyotypes of both patients were available. However, a possible translocation involving chromosome 11q13 in their tumor cells and a consecutive hMAM overexpression may be the reason for the unexpected hMAM mRNA detection in the peripheral blood of these two patients.

Additionally, in 15% to 23% of primary BC patients, the q13 region of chromosome 11 that involves the hMAM gene is amplified,^21,22 and this genetic abnormality is preferentially associated with ER-positive tumors.^23,24 Whether this association is the reason for the higher incidence of detectable hMAM mRNA in the peripheral blood of ER-positive patients compared with that of ER-negative patients is unknown, because we have not examined 11q13 amplifications in tumor samples of our BC patients. However, amplification of this chromosomal region also occurs in other human cancers, including esophageal, lung, bladder, and hepatocellular carcinoma.²⁵ It remains to be proven whether a nested RT-PCR assay for hMAM might become useful for the detection of tumor cells in peripheral blood of patients with malignancies other than BC.

We conclude that the hMAM nested RT-PCR assay may be a useful tool for the detection of circulating mammary carcinoma cells in the peripheral blood of BC patients. The clinical relevance of hMAM RT-PCR–based tumor cell detection should be further evaluated in prospective studies.

	ACKNOWLEDGMENTS

 
We thank Anneliese Kolb for excellent technical assistance and Madeleine Bohrer for critical review of the manuscript.

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Submitted June 1, 1998; accepted March 16, 1999.

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