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Published in final edited form as: Clin Chem. 2002 Aug;48(8):1225–1231.

Application of a Multigene RT-PCR assay for the detection of Mammaglobin and complementary transcribed genes in breast cancer lymph nodes

Barbara K Zehentner 1, Davin C Dillon 1, Yuqiu Jiang 1, Jiangchun Xu 1, Angela Bennington 1, David A Molesh 1, XinQun Zhang 1, Steven G Reed 1, David H Persing 1, Raymond L Houghton 1
PMCID: PMC1482782  NIHMSID: NIHMS6919  PMID: 12142378

Abstract

Background: Mammaglobin mRNA expression is found in 70-80 % of primary and metastatic breast tumor biopsies. The potential breast tumor markers B305D, B726P and GABAπ complement the expression of mammaglobin. Collectively the expression profile of these four genes could be utilized as a diagnostic and prognostic indicator for breast cancer.

Methods: A multigene RT-PCR assay was established to detect the expression of mammaglobin, GABAπ, B305D and B726P simultaneously. Specific primers and Taqman® probes were used to analyze combined mRNA expression profiles in primary breast tumors and metastatic lymph node specimens.

Results: The multigene RT-PCR assay detected significant expression signals in 27/27 primary and in 50/50 lymph-node-metastatic breast tumor samples. Specificity studies demonstrated no significant expression signal in 27 non-breast cancer lymph nodes, in 22 various normal tissues or in 14 colon tumor samples.

Conclusion: The novel RT-PCR-based assay described here provides a sensitive detection system for disseminated breast tumor cells in lymph nodes. In addition, this multigene assay could also be used to test peripheral blood and bone marrow samples.

Mammaglobin, a highly glycosylated low molecular weight protein, is a homologue of the rabbit uteroglobin and the rat steroid binding protein subunit C3 (1;2). It is a member of the Uteroglobin family with yet unknown function. The known human family members (mammaglobin, lipophilin A and B, mammaglobin B (lipophilin C, lacryglobin), Clara cell 10kDA protein) are localized in a dense cluster on chromosome 11q12.2 (3). The mRNA expression of mammaglobin has been described in 70-80 % of primary and metastatic breast tumor biopsies (1;4-7). Furthermore, mammaglobin has been used as a marker to detect circulating mammary carcinoma cells in peripheral blood using RT-PCR (8).

Although mammaglobin is a promising tumor marker it is not universally expressed in breast cancers because these tumors are highly heterogeneous by gene expression profiling. Therefore, additional markers are needed for the development of a sensitive assay to detect malignant breast cancer cells. Three genes B305D, GABAπ and B726P have been identified by genetic subtraction, differential display, DNA microarray analysis and real-time PCR, as potential therapeutic and diagnostic targets in breast cancer. Recently we reported the complementary expression profile of these candidate genes in combination with mammaglobin in breast carcinomas (7). B305D is a novel gene that is located on chromosome 21q11.1, c1 region (AP001465.1). B305D cDNA has been isolated by using differential display RT-PCR technique and its sequence predicts a type II membrane protein.

The known gene γ-aminobutyrate type A receptor π subunit (GABAπ) was recovered by PCR substraction and found to be inversely expressed as compared to B305D. GABAπ (U95367) belongs to the GABAA receptor family. Unlike other GABAA receptors that are typically expressed in neuronal tissues, GABAπ shows low expression in lung, thymus and prostate tissues and higher levels in the uterus (9). The cDNA designated as B726P (AL357148) was derived from PCR subtraction. B726P is a novel gene located on chromosome 10 with several different putative open reading frames yielded by mRNA splicing. One of these splice forms has been recently identified (10) using reactivity with autologous breast cancer patient sera and referred to as NY-BR-1.

In the present study we established a real-time multigene RT-PCR assay to simultaneously monitor the expression level of these four breast cancer markers. The sensitivity and specificity of this assay have been investigated by using numerous primary breast tumors, metastatic lymph node and normal tissues.

Materials and Methods

TISSUE SAMPLES

Primary breast cancer, normal and tumor lymph node tissue samples were kindly provided by Dr. Elizabeth Repasky (Roswell Park Cancer Institute, Buffalo, NY) and Dr. Roberto Badaro (University of Bahia, Salvador, Brazil). Tissue samples were also obtained from National Disease Research Interchange (NDRI). Lymph node cDNA samples were kindly provided by Dr. Michael Mitas (Medical University of South Carolina, Department of Surgery, Charleston SC) and Dr. Timothy P. Fleming (Washington University School of Medicine, St. Louis, MO).

RNA ISOLATION AND cDNA PREPARATION

Total RNA was extracted from liquid nitrogen frozen tissue samples by homogenization in Trizol reagent (Gibco BRL, Bethesda, MD) and cDNA was prepared using Oligo dT (Boehringer Mannheim) primer with Superscript™ II reverse transcriptase (Gibco BRL, Bethesda, MD) for 60 min at 42 °C.

PCR ASSAY

A multigene real-time PCR assay was developed to detect the expression of mammaglobin, GABAπ, B305D and B726P simultaneously. The expression levels of single genes and their combined expression levels were measured by quantitative real-time PCR using the ABI 7700 Prism™ sequence detection system (Applied Biosystems, Foster City, CA). Specific primers and 6-carboxy- fluorescein (FAM)-labeled Taqman® probes were used in combination. Primers were designed to span intron-exon junctions in order to exclude genomic DNA from amplification. Primer concentrations, limiting the reporter signal without decreasing the detected threshold cycle to avoid competition in the multigene PCR, were determined.

The forward primer for mammaglobin was 5′- tgccatagatgaattgaaggaatg-3′ and the reverse primer was 5′- tgtcatatattaattgcataaacacctca-3′, both used at 100 nM. The primer sequences for GABAπ were 5′- caattttggtggagaacccg-3′ forward and 5′- gctgtcggaggtatatggtg-3′ reverse, used at 300 nM and 50 nM respectively. For B305D amplification 300 nM of forward 5′- tctgataaaggccgtacaatg-3′ primer and 50 nM of reverse 5′- tcacgacttgctgtttttgctc-3′ primer were used.100 nM each of B726P forward 5′- gcaagtgccaatgatcagagg-3′ and reverse 5′- atatagactcaggtatacacact-3′ primer were added to the PCR mixture. Actin expression was measured in separate reactions and used to normalize expression levels. 300 nM each of forward primer 5′- actggaacggtgaaggtgaca-3′ and reverse primer 5′- cggccacattgtgaactttg-3′ were used for actin amplification. For real-time detection 4 pmol of each gene specific Taqman® probe with following sequences were used:

  • Mammaglobin FAM-5′- tcttaaccaaacggatgaaactctgagcaatg-3′-TAMRA,

  • GABAπ FAM-5′- catttcagagagtaacatggactacaca-3′-TAMRA,

  • B305D FAM-5′- atcaaaaaacaagcatggcctcacaccact-3′-TAMRA,

  • B726P FAM-5′- tcccatcagaatccaaacaagaggaagatg-3′-TAMRA,

  • Actin FAM-5′- cagtcggttggagcgagcatccc-3′TAMRA.

40 PCR cycles were performed with TaqMan® 1000 Rxn PCR Core Reagents (Part. No. 430 4439, Applied Biosystems, Foster City, CA) using 0.0375 U/μl TaqGold, 1× Buffer A, 5 mM MgCl2, 0.2 mM dCTP, 0.2 mM dATP, 0.4 mM dUTP, 0.2 mM dGTP, 0.01 U/μl AmpErase UNG, 8 % (v/v) Glycerol, 0.05 % (v/v), Gelatin, 0.01 % (v/v) Tween 20. PCR conditions were one cycle at 50°C for 2 min, one cycle at 95°C for 10 min and for 40 cycles 95°C for 15″ followed by 60°C for 1′ and 68°C for 1′. For single gene PCR standard curves were established using serial dilutions of plasmid DNA containing target gene cDNA sequence. For the multigene assay combined copy numbers were determined by using serial dilutions containing four plasmids to establish a standard curve. TaqMan® SDS analysis software was used to determine standard curves and to calculate copy numbers. Final copy numbers were determined as average values of triplicate PCR reactions and normalized per 1000 pg Actin. The average variation within triplicates was 21 %, ranging from 3 to 35 %.

RECEIVER OPERATOR CHARACTERISTIC (ROC) CURVE ANALYSIS

Rockit 0.9B Beta Version software (Dr. Charles E. Metz, Department of Radiology, The University of Chicago, http://www-radiology.uchicago.edu/krl/toppage11.htm) was used to analyze expression data obtained from pathology positive breast cancer and normal lymph node specimens. Detected copy numbers in lymph node samples for the Mammaglobin, B305D, B726P, GABAπ or the Multigene RT-PCR assay respectively were processed for the 27 actually-negative and the 50 actually- positive cases. The generated output of True-Positive Fractions (TPF) and corresponding False-Positive Fractions (FPF) was used for the construction of ROC curves as sensitivity and 1-specificity.

Results

A panel of 27 primary breast tumor samples was used to compare the multigene real-time PCR assay with single gene PCR. The multigene assay, simultaneously detecting mammaglobin + B305D + B726P + GABAπ, resulted in a significant expression signal from 27/27 tumor samples (Figure 1B) as compared to mammaglobin expression alone that detected 17 of these samples (Figure 1A). Table 1 further demonstrates the transcriptional complementation of the four target genes with B305D being highly expressed in 16, GABAπ in 10 and B726P in 14 tumor specimens. All samples tested were detected positive with a combined expression signal by the multigene assay.

Fig. 1.

Fig. 1

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Comparison of mammaglobin expression and multigene signals detected in 27 primary breast cancer tumor cDNAs. Mammaglobin expression alone was detected in 17 out of 27 breast tumors (A). The multigene assay, co-detecting mammaglobin + B305D + Gabaπ + B726P, resulted in a significant expression signal from 27 out of 27 samples (B). Multigene copy numbers were determined by establishing a standard curve using a mixture of four plasmids containing inserts of either mammaglobin, B305D, Gabaπ or B726P cDNA. Average copy numbers were determined in triplicate PCR reactions. Actin copy numbers were detected in a separate PCR assay and used for normalization.

Table 1.

Expression patternaof mammaglobin, B305D, GABAp and B726P in pimary breast tumor specimens.

breast primary tumor samples Mammaglobin B305D GABAp B726P Multigene
1 + ++ +++ +++
2 + + ++
3 ++ ++
4 ++ +++ +++ +++
5 + +++ ++
6 + +++ +++
7 + ++ ++
8 ++ +
9 ++ + +++ ++
10 ++ ++ +++ ++
11 ++ + ++
12 + ++ ++
13 +++ + +++
14 ++ + ++ +++ ++
15 + ++ ++
16 + + + ++
17 + +
18 +++ + ++
19 ++ +++ ++
20 + +
21 +++ + + +++
22 + + ++
23 + +
24 +++ + + +++
25 ++ + +
26 ++ ++
27 + + ++
Total 17/27 16/27 10/27 14/27 27/27
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a

+, <10 copies/1000pg actin; ++, <100 copies/1000pg actin; +++, <1000 copies/1000pg actin.

The expression profile of B305D and GABAπ in the breast tumor samples showed an inverse relationship which is complementary at the level of quantitative detection. This results in a detection of 24/27 primary breast tumor samples with the expression of these two genes alone. B726P expression showed an independent but additive profile in comparison to the other markers.

We then tested the cDNA of 50 pathology positive metastatic breast cancer lymph node samples and 27 normal or non-relevant disease lymph node samples respectively. All 50 metastatic breast cancer samples were detected with positive expression signals (Figure 2) whereas non-breast cancer-containing lymph node specimens showed no significant signals. Table 2 illustrates the complementary expression profiles of the four candidate genes in the 77 lymph node specimens tested.

Fig. 2.

Fig. 2

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Breast cancer lymph node analysis using the multigene Real-Time RT- PCR assay. Combined copy numbers of mammaglobin, B305D, GABAπ and B726P normalized per 1000 pg actin were detected in 50 pathology positive metastatic breast cancer lymph nodes and in 27 negative lymph nodes (24 normal + 2 melanoma + 1 lymphoma lymph node samples). Average copy numbers were determined in triplicate PCR reactions and are shown in a logarithmic format. The range of copies/1000pg Actin in metastatic breast cancer lymph nodes is 17.2 to 18574.7; and 0 - 1.6 in negative lymph nodes.

Table 2.

Positive expression signals for single gene and/or multigene analyses in lymph node specimens, in colon tumor specimens and in normal tissues.

Lymph node specimens
Metastatic breast cancer Melanoma /Lymphoma a Normal
Mammaglobin 40/50 0/3 0/24
B305D 36/50 0/3 0/24
GABAp 15/50 0/3 0/24
B726P 22/50 0/3 0/24
Multigene assay 50/50 0/3 0/24
Colon tumor specimens Normal tissues b
Multigene assay 0/14 4c /22
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a

2 melanoma and 1 lymphoma lymph node specimen.

b

2 normal colon, PBMC, bone marrow, heart, brain, pancreas, lung, liver, skin, kidney, spinal cord, salivary gland, small intestine, stomach, trachea, adrenal gland, aorta, skeletal muscle, bone, esophagus, bladder.

c

a low expression signal was detected in esophagus, skin, trachea and salivary gland cDNA (ranging from 2-6 copies/1000pg actin)

Significant mammaglobin expression alone is detected in 40 metastatic breast cancer lymph node samples, ranging from 19.7 to 4.1×10E6 copies/1000pg actin. High expression of B305D was found in 36 (5 - 5×10E3 copies/1000pg actin), of GABAπ in 15 (11.7 - 830 copies/1000pg actin) and of B726P in 22 (7 - 4.9×10E4 copies/1000pg actin) out of 50 pathology positive breast cancer lymph node samples. No significant expression signals were detected by mammaglobin, B305D, GABAπ, B726P or multigene RT-PCR in 24 normal lymph node specimens, in 2 nodes containing melanoma, or in one node with evidence of lymphoma.

The multigene real-time PCR assay detected a positive signal for all 50 metastatic breast cancer lymph node specimens, ranging from 17.2 to 1.9×10E4 copies/1000pg actin. Similar to primary tumor samples two genes in particular, B305D and GABAπ, added to the diagnostic sensitivity of mammaglobin detection in lymph node analysis. A combination of these three genes detected 50/50 lymph node breast metastases and 26/27 primary breast tumors evaluated. The inclusion of B726P enabled the detection of one primary tumor specimen. Moreover, the parallel detection of four genes enhanced the detection sensitivity for samples with low level single gene expression.

In order to assess the specificity of the multigene PCR assay described here a panel of 14 colon tumor and 22 normal tissue cDNAs was tested. Expression signals were detected in 0/14 colon tumors and in 4/22 normal tissues (Table 2). However, the low borderline signals detected in four normal tissues (esophagus, skin, trachea and salivary gland) were only ranging from 2 - 6 copies/1000pg actin, presumably due to low levels of mammaglobin expression in skin and of GABAπ and mammaglobin expression in salivary gland, trachea and esophagus. No background expression was detected in bone marrow and peripheral mononuclear cells (PBMC).

To compare the diagnostic performance of single markers and multimarker detection in lymph node analysis, we analyzed the expression data using Rockit 0.9B Beta Version software. Receiver operator characteristic (ROC) curves were established by plotting sensitivity versus 1-specificity to assess the accuracy of each assay (Figure 3). The software was used to calculate ROC curve sensitivities, specificities and the corresponding area under the curve. The area under the ROC curve (Az) indicates the performance of an assay to separate the group being tested into actually-negative and actually-positive cases. A perfect test is represented by an area of 1, an excellent test by 0.9 - 1, a good test by 0.8 - 0.9 and 0.7 - 0.9 indicates moderate accuracy. Due to perfect decision performance of the multigene assay, no actual curve could be constructed. ROC analysis of the multigene data resulted in an area value under curve of 1. The best performance for a single gene expression assay was achieved by mammaglobin, with a ROC curve area value of 0.92. B305D expression data resulted in an area value of 0.84, which indicates good test performance. B726P RT-PCR with Az = 0.78 and GABAπ RT-PCR with Az = 0.77 can be categorized as assays with moderate accuracies.

Fig. 3.

Fig. 3

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Receiver Operator Curve (ROC) analysis of Multigene and single gene Real-Time RT-PCR expression data. Expression data obtained from 27 true-negative and 50 true-positive lymph node specimens was analyzed using Rockit 0.9B Beta Version software (see Material and Methods). Sensitivity was plotted against 1-Specificity for single gene and multigene RT-PCRs. The area under each ROC curve is indicative for the diagnostic accuracy of the applied test. The maximal curve area is covered by multigene assay analysis, indicating a perfect decision performance in the sample population tested.

Discussion

Lymph node staging provides the most important prognostic indicator in breast cancer (11). The presence of metastases in lymph nodes correlates directly to the risk of disease recurrence and patient survival. Conventionally, axillary dissection is used to collect lymph nodes for analysis. As an alternative, sentinel lymphadenectomy has been discussed as a less invasive strategy to identify lymphatic spread of metastatic cancer cells (5;12;13). Nodal metastases are identified by staining (haematoxylin or eosin) or immunohistochemical analysis for cytokeratin proteins (14). Inadequate sectioning in lymph node analysis can produce false-negative results by missing small metastatic foci. Approximately 30 % of the patients diagnosed with pathologically negative lymph nodes develop recurrent disease. Since undetected micrometastases can be considered as a potential source for relapse of disease, more sensitive techniques for lymph node analysis have been discussed (5;15-17). Molecular diagnostic approaches are potentially of higher sensitivity than immunohistochemistry. However, the application of RT-PCR for breast cancer cell detection is hampered by the lack of specific marker genes. Cytokeratins as epithelial cell marker genes have shown limited specificity for breast cancer cell detection (14;18). Other potential tumor markers, in particular CEA, MUC1 and mammaglobin, demonstrated higher specificity but only identify a subset of tumor specimens. (4;5;18;19). No specific universal tumor marker has been identified so far due to the biological heterogeneity of breast carcinomas (20).

In this study we demonstrate the use of complementary expressed breast cancer genes for the development of a sensitive and specific RT-PCR assay to detect metastatic breast cancer cells. The multimarker assay described here increases the probability of detecting disseminated tumor cells and also provides the potential to characterize micrometastases on a molecular level. This could lead to improvements in determination of prognosis, monitoring of disease and possibly to individualized therapeutic strategies in the future.

For the future, intraoperative analysis of sentinel lymph nodes based on RT-PCR might even be possible. Due to improved screening approaches, early detection of breast tumors with small diameters is increasing. Since the incidence of lymph node metastases is related to tumor size, for small tumors the value of routine axillary lymph node dissection, in consideration of the possibility of overtreatment and consequent morbidity, is a matter of much debate (21-23). The development of rapid quantitative RT-PCR protocols might provide the platform to realize highly sensitive intraoperative analysis of sentinel lymph nodes (24) and to gain prognostic information about the necessity of axillary dissection for disease control.

This study focused on lymph node analysis to evaluate specificity and sensitivity of the assay. However, the multigene assay has potential applications in various tissues as a sensitive tool to detect breast tumor cells. Recently, we reported initial studies for these four genes individually (7) and in combination (25) to detect circulating breast tumor cells in peripheral blood samples.

Metastatic cells in bone marrow are also discussed to provide a high predictive value (26) for relapse of disease, independent of lymph node status. In addition, cytokeratin positive cells detected in the bone marrow of early stage tumors are suggesting a potential for early diagnosis of tumor dissemination (27). RT-PCR for mammaglobin has been shown to successfully detect bone marrow micrometastases (28). Mammaglobin gene expression is also a useful marker for detection of tumor cell contamination in leukaphereses, with potential application in autologous stem cell transplantation protocols for high-dose chemotherapy programs (29). Due to the increased sensitivity provided by the multigene assay compared to mammaglobin alone, we conclude that the method described here will be useful for the evaluation of peripheral blood, bone marrow and leukapheresis samples for disseminated breast cancer cells.

Acknowledgement:

This work was supported in part by NIH grants CA-75794 and CA-86673. We would like to acknowledge Dr. Roberto Badaro (University of Bahia, Salvador, Brazil), and Dr. Marcos Nolasco and Dr. Aristides Maltez Filho from the Hospital Aristides Maltez (Salvador, Brazil) for kindly providing us primary breast tumor tissues. We also want to thank Dr. Elizabeth Repasky (Roswell Park Cancer Institute, Buffalo, NY) for providing tissue samples and Dr. Michael Mitas (Medical University of South Carolina, Department of Surgery, Charleston SC) and Dr. Timothy P. Fleming (Washington University School of Medicine, St. Louis, MO) for providing lymph node cDNA samples.

Footnotes

Abbreviations:
RT-PCR
reverse transcription-PCR;
GABAπ
γ-aminobutyrate type A receptor π subunit;
CEA
carcinoembryonic antigen;
MUC1
mucin1;
NDRI
National Disease Research Interchange
FAM
6-carboxy-fluorescein.
ROC
receiver operator characteristic

References:

  • 1.Watson MA, Fleming TP. Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer. Cancer Res. 1996;56:860–5. [PubMed] [Google Scholar]
  • 2.Watson MA, Darrow C, Zimonjic DB, Popescu NC, Fleming TP. 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;16:817–24. doi: 10.1038/sj.onc.1201597. [DOI] [PubMed] [Google Scholar]
  • 3.Ni J, Kalff-Suske M, Gentz R, Schageman J, Beato M, Klug J. All human genes of the uteroglobin family are localized on chromosome 11q12.2 and form a dense cluster. Ann N Y Acad Sci. 2000;923:25–42. doi: 10.1111/j.1749-6632.2000.tb05517.x. [DOI] [PubMed] [Google Scholar]
  • 4.Leygue E, Snell L, Dotzlaw H, Hole K, Troup S, Hiller-Hitchcock T, et al. Mammaglobin, a potential marker of breast cancer nodal metastasis. J Pathol. 1999;189:28–33. doi: 10.1002/(SICI)1096-9896(199909)189:1<28::AID-PATH389>3.0.CO;2-H. [DOI] [PubMed] [Google Scholar]
  • 5.Min CJ, Tafra L, Verbanac KM. Identification of superior markers for polymerase chain reaction detection of breast cancer metastases in sentinel lymph nodes. Cancer Research. 1998;58:4581–4. [PubMed] [Google Scholar]
  • 6.Watson MA, Dintzis S, Darrow CM, Voss LE, Jensen R, Fleming TP. Mammaglobin expression in primary, metastatic, and occult breast cancer. Cancer Reserch. 1999;59:3028–31. [PubMed] [Google Scholar]
  • 7.Houghton RL, Dillon DC, Molesh DA, Zehentner BK, Xu JC, Jiang J, et al. Transcriptional Complementarity in Breast Cancer: Application to Detection of Circulating Tumor Cells. Mol Diagn. 2001;6:79–91. doi: 10.1007/BF03262038. [DOI] [PubMed] [Google Scholar]
  • 8.Zach O, Kasparu H, Krieger O, Hehenwarter W, Girschikofsky M, Lutz D. Detection of circulating mammary carcinoma cells in the peripheral blood of breast cancer patients via a nested reverse transciptase polymerase chain reaction for mammaglobin mRNA. J Clin Oncol. 1999;17:2015–9. doi: 10.1200/JCO.1999.17.7.2015. [DOI] [PubMed] [Google Scholar]
  • 9.Hedblom E, Kirkness EF. A novel class of GABAA receptor subunit in tissues of the reproductive system. J Biol Chem. 1997;272:15346–50. doi: 10.1074/jbc.272.24.15346. [DOI] [PubMed] [Google Scholar]
  • 10.Jager D, Stockert E, Gure AO, Scanlan MJ, Karbach J, et al. Identification of a tissue-specific putative transcription factor in breast tissue by serological screening of a breast cancer library. Cancer Research. 2001;61:2055–61. [PubMed] [Google Scholar]
  • 11.Donegan WL, Spratt JS, editors. Gross and Microscopic Pathology. W.B. Saunders Company; Cancer of the Breast. Philadelphia: 1995. pp. 267–9. [Google Scholar]
  • 12.Noguchi M, Tsugawa K, Bando E, Kawahara F, Miwa K, Yokoyama K, et al. Sentinel lymphadenectomy in breast cancer: identification of sentinel lymph node and detection of metastases. Breast Canc Res Treat. 1999;53:97–104. doi: 10.1023/a:1006118827167. [DOI] [PubMed] [Google Scholar]
  • 13.Whitworth P, McMasters KM, Tafra L, Edwards MJ. State-of-the-art lymph node staging for breast cancer in the year 2000. Am J Surg. 2000;180:262–7. doi: 10.1016/s0002-9610(00)00465-7. [DOI] [PubMed] [Google Scholar]
  • 14.Kostler WJ, Brodowicz T, Hejna M, Wiltschke C, Zielinski C. Detection of minimal residual disease in patients with cancer: a review of techniques, clinical implications, and emerging therapeutic consequences. Cancer Detect Prev. 2000;24:376–403. [PubMed] [Google Scholar]
  • 15.Mori M, Mimori K, Inoue H, Barnard GF, Tsuji K, Nanbara S, et al. Detection of cancer micrometastases in lymph nodes by reverse transcriptase- polymerase chain reaction. Cancer Research. 1995;55:3417–20. [PubMed] [Google Scholar]
  • 16.Ooka M, Sakita I, Fujiwara Y, Tamaki Y, Yamamoto H, Aihara T, et al. Selection of mRNA markers for detection of lymph node micrometastases in breast cancer patients. Oncology Rep. 2000;7:561–6. doi: 10.3892/or.7.3.561. [DOI] [PubMed] [Google Scholar]
  • 17.Pantel K, von Knebel Doeberitz M. Detection and clinical relevance of micrometastatic cancer cells. Curr Opin Oncol. 2000;12:95–101. doi: 10.1097/00001622-200001000-00016. [DOI] [PubMed] [Google Scholar]
  • 18.Zippelius A, Pantel K. RT-PCR-based detection of occult disseminated tumor cells in peripheral blood and bone marrow of patients with solid tumors. Ann N Y Acad Sci. 2000;906:110–23. doi: 10.1111/j.1749-6632.2000.tb06600.x. [DOI] [PubMed] [Google Scholar]
  • 19.Mitas M, Mikhitarian K, Walters C, Baron PL, Elliott BM, Brothers TE, et al. Quantitative real-time RT-PCR detection of breast cancer micrometastasis using a multigene marker panel. Int J Cancer. 2001;93:162–71. doi: 10.1002/ijc.1312. [DOI] [PubMed] [Google Scholar]
  • 20.Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, et al. Molecular portraits of human breast tumours. Nature. 2000;406:747–52. doi: 10.1038/35021093. [DOI] [PubMed] [Google Scholar]
  • 21.Sakorafas GH, Tsiotou AG, Balsiger BM. Axillary lymph node dissection in breast cancer. Acta Oncol. 2000;39:455–66. doi: 10.1080/028418600750013366. [DOI] [PubMed] [Google Scholar]
  • 22.Lindahl T, Engel G, Ahlgren J, Klaar S, Bjohle J, Lindman H, et al. Can axillary dissection be avoided by improved molecular biological diagnosis? Acta Oncol. 2000;39:319–26. doi: 10.1080/028418600750013087. [DOI] [PubMed] [Google Scholar]
  • 23.Cserni G. Axillary staging of breast cancer and the sentinel node. J Clin Pathol. 2000;53:733–41. doi: 10.1136/jcp.53.10.733. [DOI] [PMC free article] [PubMed] [Google Scholar]
  • 24.Godfrey TE, Raja S, Hughes S, Finkelstein SD, Luketich JD. Detection of occult micrometstases using intraoperative, quantitative RT-PCR [Abstract]. Clin Chem. 2001;47:A103. [Google Scholar]
  • 25.Zehentner BK, Dillon DC, Jiang J, Xu JC, Badaro R, Zhang X, et al. A Multigene real-time PCR assay for the detection of complementary transcribed genes in breast cancer [Abstract]. Clin Chem. 2001;47:A129. [PMC free article] [PubMed] [Google Scholar]
  • 26.Janni W, Hepp F, Rjosk D, Kentenich C, Strobl B, Schindlbeck C, et al. The fate and prognostic value of occult metastatic cells in the bone marrow of patients with breast carcinoma between primary treatment and recurrence. Cancer. 2001;92:46–53. doi: 10.1002/1097-0142(20010701)92:1<46::aid-cncr1290>3.0.co;2-6. [DOI] [PubMed] [Google Scholar]
  • 27.Braun S, Pantel K, Muller P, Janni W, Hepp F, Kentenich CR, et al. Cytokeratin-positive cells in the bone marrow and survival of patients with stage I, II, or II breast cancer. N Engl J Med. 2000;342:525–33. doi: 10.1056/NEJM200002243420801. [DOI] [PubMed] [Google Scholar]
  • 28.Ooka M, Tamaki Y, Sakita I, Fujiwara Y, Yamamoto H, Miyake Y, et al. Bone marrow micrometastases detected by RT-PCR for mammaglobin can be an alternative prognostic factor of breast cancer. Breast Cancer Res Treat. 2001;67:169–75. doi: 10.1023/a:1010651632354. [DOI] [PubMed] [Google Scholar]
  • 29.Leone F, Perissinotto E, Viale A, Cavalloni G, Taraglio S, Capaldi A, et al. Detection of breast cancer cell contamination in leukapheresis product by real-time quantitative polymerase chain reaction. Bone Marrow Transplant. 2001;27:517–23. doi: 10.1038/sj.bmt.1702815. [DOI] [PubMed] [Google Scholar]

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