Centro de Investigação de Patobiologia Molecular (CIPM), Instituto Português de Oncologia de Francisco Gentil, Lisboa, Portugal
Received 28 May 2001; revised 1 October 2001; accepted 23 October 2001.
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Abstract |
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The aim of this study was: (i) to evaluate the sensitivity and specificity of mammaglobin as a reverse transcriptase polymerase chain reaction (RT-PCR) marker of breast cancer cells; (ii) to determine the incidence of tumor cell contamination of hematopoietic samples from patients with breast cancer.
Materials and methods
A nested RT-PCR assay for mammaglobin was developed. Sensitivity was determined by serial dilution assays with breast cancer cell lines, human breast cancers and normal breast tissue. Specificity was evaluated in hematopoietic samples from healthy volunteers and patients with hematological malignancies or solid tumors other than breast cancer.
Results
The mammaglobin transcript was detected in all 15 breast cancers, one benign breast tumor and five normal breast tissues studied, as well as in three breast cancer cell lines, in dilutions as low as 108. The transcript was not detected in any of 47 peripheral blood samples, 15 bone marrow aspirates and 28 peripheral blood progenitor cell samples from the three control populations. Mammaglobin mRNA was detected in 19 of 78 peripheral blood samples from patients with breast cancer starting systemic chemotherapy, as well as in five of 30 repeat samples collected before the fourth cycle of treatment. The transcript was also present in six of seven bone marrow aspirates from patients with metastatic disease, two of five with loco-regional disease, but not in the aspirate of two patients with thrombocytopenia and a previous history of breast cancer.
Conclusions
Human mammaglobin mRNA is a sensitive and specific marker of breast cancer cells and should be further studied as a molecular marker of tumor cell contamination of hematopoietic tissues.
Key words: breast cancer, mammaglobin, minimal residual disease
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Introduction |
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In contrast to hematological malignancies, solid tumors rarely have specific diagnostic genetic changes. To overcome this limitation, tissue-specific markers have been widely evaluated as potential molecular targets for the detection of occult tumor cells [46]. A variety of tissue specific markers, including different cytokeratin transcripts, have been widely evaluated as targets for detection of occult breast cancer cells by reverse transcriptase polymerase chain reaction (RT-PCR), but have been shown to be non-specific [711].
In contrast to cytokeratins, human mammaglobin, a member of the uteroglobin gene family, was reported to be exclusively expressed in mammary epithelium and overexpressed in some breast cancers, making it a potentially useful RT-PCR target for breast cancer cell detection in hematopoietic products [1213]. Thus, we developed a nested RT-PCR strategy to detect the mammaglobin transcript, and evaluated the sensitivity and specificity of this molecular marker for detection of minimal residual disease in breast cancer patients. The sensitivity was determined by dilution tests using both tumor cells from human breast cancer cell lines and total RNA obtained from human breast cancer cell lines and fresh tumors. The specificity of the assay was evaluated using as negative controls for mammaglobin expression hematopoietic tumor cell lines and hematopoietic samples from healthy volunteers, from patients with hematological malignancies and from patients with solid tumors other than breast cancer. Once the specificity and sensitivity of the marker were established, we then assessed the incidence of mammaglobin expression in bone marrow, peripheral blood and peripheral blood progenitor cell (PBPC) samples collected from breast cancer patients.
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Materials and methods |
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Human cancer cell lines and tissue samples
The human breast cancer cell lines MCF-7, ZR75.1 and T47D were used. These cell lines were cultured in minimal essential medium (MEM; Sigma-Aldrich, UK), Dulbeccos modified eagles medium (DMEM; Sigma-Aldrich) and RPMI-1640 (Gibco-BRL, USA) media, respectively, supplemented with 10% fetal bovine serum (FBS; Gibco-BRL), 4 mM of L-glutamin (Gibco-BRL), 50 U/ml of penicillin (Gibco-BRL) and 5 µg/ml of streptomycin (Gibco-BRL). Cultures were routinely fed every 3 days by splitting into one to two subcultures.
Nine hematopoietic cell lines (NB-4, K562, HL-60, BV173, TOM-1, MV-4.11, Kassumi, ME-1 and Sup-B27) were also used in this study. All these cell lines were grown in RPMI-1640 medium supplemented with 10% FBS, L-glutamin (4 mM), penicillin (50 U/ml) and streptomycin (5 µg/ml).
Samples from 15 breast cancers (14 from previously untreated patients), one benign tumor and five normal breast tissues were analyzed. The tissue samples were removed from the surgical specimen or obtained by biopsy, immediately frozen and stored in liquid nitrogen before analysis.
Hematopoietic samples
Peripheral blood samples were obtained from 78 patients with breast cancer (median age 54 years; range 2875 years). In the 65 patients with loco-regional disease, the samples were collected prior to initiation of adjuvant chemotherapy; in the remaining 13 patients who had metastatic disease, the sample was collected before initiation of first line chemotherapy. Additionally, in 30 of these 78 patients, a second peripheral blood sample was collected before the administration of the fourth cycle of cytotoxic chemotherapy. Bone marrow aspirates and PBPC samples from 14 and two breast cancer patients, respectively, were also studied.
Hematopoietic samples from 43 patients with hematological malignancies (peripheral blood, 17; bone marrow, 11; PBPC, 15), from 14 patients with solid tumors other than breast cancer (peripheral blood, seven; bone marrow, two; PBPC, five) and from 33 healthy volunteers (peripheral blood, 23; bone marrow, two; PBPC, eight) were used as negative controls for the detection of mammaglobin expression.
Nucleated cells from peripheral blood (5 ml), bone marrow (3 ml) and PBPC (1 ml) samples were isolated by an osmotic red blood cell lysis buffer (155 mM NH4Cl; 10 mM KHCO3; 0.1 mM EDTA; pH 7.4). The pellets were then stored at 70°C.
RNA extraction
Total cellular RNA was extracted from cell lines, tissue samples and nucleated cells of peripheral blood, bone marrow and PBPC using TRIZOL reagent (Gibco-BRL) according to the manufacturers instructions. Quantification and purity assessment was performed by optical density (OD) measurement at 260 and 280 nm.
RT-PCR
cDNAs were reverse transcribed from 1.5 µg of total RNA in a 20 µl reaction. Briefly, the RNA, 3 µg of random hexamers and an adequate volume of DEPC-treated water to make up a total volume of 8.5 µl were heated to 72°C for 10 min. Then, 11.5 µl of the RT mixture containing 4 µl of 5 x First Strand Buffer (250 mM TrisHCl pH 8.3, 375 mM KCl, 15 mM MgCl2; Gibco-BRL), 10 pmol of dNTPs (Gibco-BRL), 0.2 pmol of DTT (Gibco-BRL), 20 U of RNAase inhibitor (RNase OutTM, Gibco-BRL) and 200 U of reverse transcriptase (SuperscriptTM; Gibco-BRL) were added. The final mixture was incubated at 37°C for 1.5 h, followed by a final incubation at 95°C for 5 min.
PCR reactions were performed in a 50 µl reaction containing 2.5 µl of the synthesized cDNA, 5 µl of 10 x PCR buffer (200 mM TrisHCl pH 8.4, 500 mM KCl) (Gibco-BRL), 10 pmol dNTPs, 1 U Taq DNA polymerase (Gibco-BRL), 20 pmol of primers and 1.25 mM of MgCl2. The PCR conditions were set up as follows: initial denaturation at 95°C for 3 min, 35 cycles at 95°C for 30 s (denaturation), 58°C for 1 min (annealing) and 72°C for 1 min (extension), followed by a final extension at 72°C for 10 min. One microliter of the first PCR products was subjected to a second amplification by using a set of internal primers and conditions identical to the first PCR round with the exception of the MgCl2 concentration (1.5 mM). The nested mammaglobin-specific oligonucleotide primers [14] were located at different exons to avoid the amplification of genomic DNA, giving origin to fragments of 325 and 202 bp in the first and second PCR amplification round, respectively. The PCR products were analyzed in a 2% agarose ethidium bromide-stained gel. In order to confirm the nucleotide sequence of the amplified products, purified nested PCR products were directly sequenced using SequenaseTM version 2.0 DNA sequencing kit (Amersham Life Science, USA).
Each patient sample was tested twice and considered to be positive if a specific amplification was detected in at least one PCR assay. To reduce the risk of contamination, different rooms were used for RNA extraction, for first and second step PCR and for analysis of RT-PCR products. Water negative controls were included in each PCR experiment to test the contamination of both RT and PCR components: RT-PCR control containing all components of the reaction but no target RNA; first step PCR control performed without template cDNA; and second step PCR control performed using the first PCR negative control sample as the template. As positive controls, cDNA from breast cancer cell lines or patient tumors, as well as cDNA samples of diluted breast cancer RNA corresponding to the established RT-PCR limit of sensitivity, were also included. The quality of the isolated RNA and adequate cDNA synthesis was confirmed by cDNA amplification of the bcr gene that is expressed in all cell types [15].
Sensitivity of RT-PCR
The sensitivity of the RT-PCR assay for the detection of mammaglobin mRNA was established by serially diluting total RNA from breast cancer cell lines and patient tumors into Escherichia coli tRNA, in concentrations ranging from 1 to 108 µg of mammaglobin-positive RNA in a total of 1 µg RNA. These dilutions were then submitted to the RT-PCR assay.
Cell dilution experiments were also performed to test the potential sensitivity of the technique in the clinical setting. In these experiments, tumor cells from each breast cancer cell line were serially diluted in mammaglobin-negative PBPC, in concentrations ranging from 106 to 10 breast cancer cells in a total of 107 cells.
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Results |
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Bone marrow aspirates from 14 patients with a diagnosis of breast cancer were also studied (Table 1). Twelve patients had clinical evidence of disease: seven had documented metastases and five were undergoing adjuvant chemotherapy for recently diagnosed breast cancer. The two other patients had been diagnosed and treated for breast cancer 4 and 10 years earlier, respectively, and had a bone marrow biopsy performed for evaluation of (transient) thrombocytopenia. Mammaglobin mRNA was reproducibly detected with both PCR tests in six of seven patients with metastatic disease, and in two of five patients with loco-regional disease. In the two other patients with a past diagnosis of breast cancer the mammaglobin RNA transcript was not detected. The histological evaluation of the bone marrow biopsy specimen detected involvement in only four of these six patients with known metastatic disease (Figure 3). In one patient treated with systemic chemotherapy for widespread metastatic disease (including bone involvement), the mammaglobin transcript was not detected, and neither was there evidence of histological involvement in the bone marrow biopsy. The two patients with loco-regional disease who had mammaglobin RNA-positive bone marrow aspirates had both stage II disease with one and 17 positive axillary lymph nodes, respectively.
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Discussion |
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In most solid tumors, the lack of cancer-specific genetic markers led to the study of tissue-specific markers to identify contaminating tumor cells in hematopoietic tissues. In breast cancer, cytokeratins have been extensively studied but several groups documented low specificity of these markers [711]. This is probably due to a low level of illegitimate expression of those genes in hematopoietic cells [20]. In contrast, with a nested RT-PCR strategy, we documented that the identification of human mammaglobin mRNA is both a specific and a sensitive marker of breast cancer cells. The use of primers spanning more than one exon generated a cDNA-specific band in the gel, avoiding a decrease of specificity from any genomic DNA contamination. Indeed, the transcript was not present in almost one hundred peripheral blood, bone marrow or PBPC samples from healthy volunteers or from patients with hematological malignancies or solid tumors other than breast cancer, also suggesting no evidence for the presence of human mammaglobin pseudogenes. In contrast, the human mammaglobin mRNA was detected in all breast cancers tested as well as in normal breast tissue and in a benign breast tumor, documenting tissue specificity. These results confirm the high frequency of mammaglobin expression in human breast cancers previously reported [12, 13]. Furthermore, in serial dilution assays the marker was usually detected in concentrations as low as 106, compatible with a high level of expression of the mammaglobin gene in breast tissue. Nevertheless, in one breast cancer sample obtained from a patient, as well as with the ZR75.1 cell line, the limit of detection was only 101, suggesting that the level of mammaglobin expression varies between breast cancers. The apparent discrepancy between a large PCR band at the 101 dilution and the absence of band at 102 is most likely the result of the large number of cycles (35) used in both PCR rounds, resulting in a high amount of production in the second amplification step, even when starting with a small amount of the transcript in the initial step. These experiments were repeated with identical results.
Overall, 22% (24 of 110) of the peripheral blood samples collected from patients with breast cancer were positive for the human mammaglobin transcript. Seventy-eight of these samples (with 24% positivity for the transcript) were collected before the initiation of adjuvant chemotherapy or of first- line chemotherapy for metastatic disease. This is in contrast to a low incidence of breast cancer cell contamination of peripheral blood detected by immunocytochemistry in other series [21, 22]. The trend towards a higher incidence of mammaglobin-positive samples from stage I (0/10), to stages IIIII (14/55), to stage IV breast cancer (5/13) should be proportional to increasing patient tumor burdens, as previously reported by Zach et al. [14]. However, in this series there was an unexplained decrease in the incidence of contamination between stage II and stage III patients: 28% (11/39) and 19% (3/16), respectively (P = 0.7 using Fishers exact test), but the sample size is too small for any meaningful clinical interpretation. Whether peripheral blood cell separation using pre-enrichment methods would increase the sensitivity of human mammaglobin mRNA detection by PCR is not known. However, the high cost of pre-enrichment methods may limit the clinical applicability of assays used to study minimal residual disease, even if the detection of residual tumor cells is shown to be clinically relevant.
The incidence of mammaglobin mRNA-positive bone marrow aspirates was 57% (8/14). This higher incidence of transcript detection in bone marrow aspirates compared with peripheral blood samples most likely reflect that seven of the 14 aspirates were obtained from patients with breast cancer and bone metastases. Still, in only four of the six patients with detection of the human mammaglobin mRNA in the aspirate was there histological evidence of tumor involvement in the bone marrow biopsy. In no cases was there histological evidence of tumor involvement without a positive aspirate for human mammaglobin mRNA.
All RT-PCR tests were done in duplicate. While positive bone marrow aspirates were positive in both tests, for the majority of positive peripheral blood samples the transcript was identified in only one of the two tests. This most likely reflects a sampling effect due to a lower number of mammaglobin-positive cells in the peripheral blood compared with bone marrow. Indeed, the transcript could not be detected in peripheral blood samples if processed with a 1:10 dilution. It has been reported that when the levels of the molecular target are near the detection limit of the RT-PCR technique, false negative results can occur [23].
In the subgroup of 30 patients with paired pre-chemotherapy and on-chemotherapy peripheral blood samples studied, there was no clear pattern of tumor involvement related to the administration of chemotherapy. While there were patients with a positive pre-treatment and a negative on-treatment sample, suggesting in vivo tumor purging associated with the administration of the cytotoxic treatment, there were also patients with a negative pre-treatment and a positive on-treatment sample. This latter observation could be attributed to tumor cell mobilization, as described by Brugger et al. [24], but most likely reflect a sampling effect at the lower limit of the detection for the RT-PCR assay. Indeed, the analysis of the distribution of discordant pairs, pre-chemotherapy positive/post-chemotherapy negative (4) and pre-chemotherapy negative/post-chemotherapy positive (3), shows no statis-tically significant differences (P >0.05 using McNemars test).
Immunocytochemistry, most commonly targeting cytokeratin expression, is still regarded as the gold standard technique for evaluation of minimal residual disease in breast cancer patients. This methodology was not used in this study as a control. However, the purpose of this study was not to compare human mammaglobin mRNA detection by PCR with cytokeratin immunocytochemistry as different methods of evaluation of minimal residual disease, but to document the sensitivity and the specificity of the human mammaglobin RT-PCR assay and to obtain exploratory clinical data.
In conclusion, the detection of human mammaglobin mRNA by RT-PCR is a sensitive and specific marker of breast cancer cells and should be studied further as a molecular marker of tumor contamination of hematopoietic tissues.
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Acknowledgements |
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Footnotes |
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References |
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![]() ![]() ![]() ![]() ![]() ![]() ![]() |
---|
2.
Diel IJ, Kaufmann M, Costa SD et al. Micrometastatic breast cancer cells in bone marrow at primary surgery: prognostic value in comparison with nodal status. J Natl Cancer Inst 1996; 88: 16521658.
3.
Pantel K, Cote RJ, Fodstad O. Detection and clinical importance of micrometastatic disease. J Natl Cancer Inst 1999; 91: 11131124.
4. Pantel K. Detection of minimal disease in patients with solid tumors. J Hematother 1996; 5: 359367.[Medline]
5. Raj GV, Moreno JG, Gomella LG. Utilization of polymerase chain reaction technology in the detection of solid tumors. Cancer 1998; 82: 14191442.[ISI][Medline]
6. Lambrechts AC, vant Veer LJ, Rodenhuis S. The detection of minimal numbers of contaminating epithelial tumor cells in blood or bone marrow: use, limitations and future of RNA-based methods. Ann Oncol 1998; 9: 12691276.[Abstract]
7. Burchill SA, Bradbury MF, Pittman K et al. Detection of epithelial cancer cells in peripheral blood by reverse transcriptase-polymerase chain reaction. Br J Cancer 1995; 71: 278281.[ISI][Medline]
8. Bostick PJ, Chatterjee S, Chi DD et al. Limitations of specific reverse-transcriptase polymerase chain reaction markers in the detection of metastases in the lymph nodes and blood of breast cancer patients. J Clin Oncol 1998; 16: 26322640.[Abstract]
9. Jung R, Petersen K, Krüger W et al. Detection of micrometastasis by cytokeratin 20 RT-PCR is limited due to stable background transcription in granulocytes. Br J Cancer 1999; 81: 870873.[ISI][Medline]
10. Champelovier P, Mongelard F, Seigneurin D. CK20 gene expression: technical limits for the detection of circulating tumor cells. Anticancer Res 1999; 19: 20732078.[ISI][Medline]
11. Silva AL, Diamond J, Silva MR, Passos-Coelho JL. Cytoketatin 20 is not a reliable molecular marker for occult breast cancer cell detection in hematological tissues. Breast Cancer Res Treat 2001; 66: 5966.[ISI][Medline]
12. Watson MA, Fleming TP. Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Res 1996; 56: 860865.[Abstract]
13.
Watson MA, Dintzis S, Darrow CM et al. Mammaglobin expression in primary, metastatic, and occult breast cancer. Cancer Res 1999; 59: 30283031.
14.
Zach O, Kasparu H, Krieger O et al. 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. J Clin Oncol 1999; 17: 20152019.
15.
Diamond J, Goldman JM, Melo JV. BCR-ABL, ABL-BCR, BCR and ABL genes are all expressed in individual granulocyte-macrophage colony-forming unit colonies derived from blood of patients with chronic myeloid leukemia. Blood 1995; 85: 21712215.
16. Watson MA, Darrow C, Zimonjic DB et al. Structure and transcriptional regulation of the human mammaglobin gene, a breast cancer associated member of the uteroglobin gene family localized to chromosome 11q13. Oncogene 1998; 16: 817824.[ISI][Medline]
17. Fields KK, Elfenbein GJ, Trudeau WL et al. Clinical significance of bone marrow metastases as detected using the polymerase chain reaction in patients with breast cancer undergoing high-dose chemotherapy and autologous bone marrow transplantation. J Clin Oncol 1996; 14: 18681876.[Abstract]
18.
van Rhee F, Lin F, Cullis JO et al. Relapse of chronic myeloid leukemia after allogenic bone marrow transplant: the case for giving donor leukocyte transfusions before the onset of hematologic relapse. Blood 1994; 83: 33773383.
19.
Diverio D, Rossi V, Avvisati G et al. Early detection of relapse by prospective reverse transcriptase-polymerase chain reaction analysis of the PML/RAR fusion gene in patients with acute promyelocytic leukemia enroled in the GIMEMA-AIEOP multicenter AIDA trial. Blood 1998; 92: 784789.
20. Zippelius A, Kufer P, Honold G et al. Limitations of reverse-transcriptase polymerase chain reaction analyses for detection of micrometastatic epithelial cancer cells in bone marrow. J Clin Oncol 1997; 15: 27012708.[Abstract]
21.
Passos-Coelho JL, Ross AA, Moss TJ et al. Absence of breast cancer cells in a single-day peripheral blood progenitor cell collection after priming with cyclophosphamide and granulocyte-macrophage colony-stimulating factor. Blood 1995; 85: 11381143.
22. Passos-Coelho JL, Ross AA, Kahn DJ et al. Similar breast cancer cell contamination of single-day peripheral-blood progenitor-cell collections obtained after priming with hematopoietic growth factor alone or after cyclophosphamide followed by growth factor. J Clin Oncol 1996; 14: 25692575.[Abstract]
23. Melo JV, Yan XH, Diamond J et al. Reverse transcription/polymerase chain reaction (RT/PCR) amplification of a very small number of transcripts: the risk in misinterpreting negative results. Leukemia 1996; 10: 12171221.[ISI][Medline]
24.
Brugger W, Bross KJ, Glatt M et al. Mobilization of tumor cells and hematopoietic progenitor cells into peripheral blood of patients with solid tumors. Blood 1994; 83: 636640.