The online version of this article (doi:10.1186/bcr2922) contains supplementary material, which is available to authorized users.
The authors declare that they have no competing interests.
ST planned and performed all experiments. RK arranged the collection of clinical material. ST, KA and OF participated in the design of the study, discussion and interpretation of results. ST drafted the manuscript and OF revised it. All authors read and approved the final manuscript.
The presence of tumor cells in the axillary lymph nodes is the most important prognostic factor in early stage breast cancer. However, the optimal method for sentinel lymph node (SLN) examination is still sought and currently many different protocols are employed. To examine two approaches for tumor cell detection we performed, in sequence, immunomagnetic enrichment and RT-PCR analysis on SLN samples from early stage breast cancer patients. This allowed us to compare findings based on the expression of cell surface proteins with those based on detection of intracellular transcripts.
Enrichment of EpCAM and Mucin 1 expressing cells from fresh SLN samples was achieved using magnetic beads coated with the appropriate antibodies. All resulting cell fractions were analyzed by RT-PCR using four chosen breast epithelial markers (hMAM, AGR2, SBEM, TFF1). Gene expression was further analyzed using RT-PCR arrays and markers for epithelial to mesenchymal transition (EMT).
Both EpCAM and Mucin 1 enriched for the epithelial-marker expressing cells. However, EpCAM-IMS identified epithelial cells in 71 SLNs, whereas only 35 samples were positive with RT-PCR targeting breast epithelial transcripts. Further analysis of EpCAM positive but RT-PCR negative cell fractions showed that they had increased expression of MMPs, repressors of E-cadherin, SPARC and vimentin, all transcripts associated with the process of epithelial to mesenchymal transition.
The EpCAM IMS-assay detected tumor cells with epithelial and mesenchymal-like characteristics, thus proving to be a more robust marker than pure epithelial derived biomarkers. This finding has clinical implications, as most methods for SLN analysis today rely on the detection of epithelial transcripts or proteins.
Additional file 1: Primers and probes used for RT-PCR. Sequence of primers designed using ProbeFinder from Roche Applied Science and the corresponding probes from the Universal ProbeLibrary [ 28](DOC 53 KB)
Additional file 2: SLN samples used on the EMT-arrays and EMT RT-PCR with calculations. A complete overview of all clinical samples used in this study. The number of Immunomagnetic selected (IMS) positive cells per sample and the hMAM status as determined by RT-PCR is listed. Then the EMT-array RT-PCR data are shown, both raw Ct values and the normalized expression data. Following this is the hierarchical clustering analysis and the different t-tests. (XLS 214 KB)13058_2010_2730_MOESM2_ESM.XLS
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Fisher B, Bauer M, Wickerham DL, Redmond CK, Fisher ER, Cruz AB, Foster R, Gardner B, Lerner H, Margolese R, et al: Relation of number of positive axillary nodes to the prognosis of patients with primary breast cancer. An NSABP update. Cancer. 1983, 52: 1551-1557. 10.1002/1097-0142(19831101)52:9<1551::AID-CNCR2820520902>3.0.CO;2-3. CrossRefPubMed
Fitzgibbons PL, Page DL, Weaver D, Thor AD, Allred DC, Clark GM, Ruby SG, O'Malley F, Simpson JF, Connolly JL, Hayes DF, Edge SB, Lichter A, Schnitt SJ: Prognostic factors in breast cancer. College of American Pathologists Consensus Statement 1999. Arch Pathol Lab Med. 2000, 124: 966-978. PubMed
Giuliano AE, Jones RC, Brennan M, Statman R: Sentinel lymphadenectomy in breast cancer. J Clin Oncol. 1997, 15: 2345-2350. PubMed
Cote RJ, Peterson HF, Chaiwun B, Gelber RD, Goldhirsch A, Castiglione-Gertsch M, Gusterson B, Neville AM: Role of immunohistochemical detection of lymph-node metastases in management of breast cancer. International Breast Cancer Study Group. Lancet. 1999, 354: 896-900. 10.1016/S0140-6736(98)11104-2. CrossRefPubMed
de Boer M, van Deurzen CH, van Dijck JA, Borm GF, van Diest PJ, Adang EM, Nortier JW, Rutgers EJ, Seynaeve C, Menke-Pluymers MB, Bult P, Tjan-Heijnen VC: Micrometastases or isolated tumor cells and the outcome of breast cancer. New Engl J Med. 2009, 361: 653-663. 10.1056/NEJMoa0904832. CrossRefPubMed
Reed J, Rosman M, Verbanac KM, Mannie A, Cheng Z, Tafra L: Prognostic implications of isolated tumor cells and micrometastases in sentinel nodes of patients with invasive breast cancer: 10-year analysis of patients enrolled in the prospective East Carolina University/Anne Arundel Medical Center Sentinel Node Multicenter Study. J Am Coll Surg. 2009, 208: 333-340. 10.1016/j.jamcollsurg.2008.10.036. CrossRefPubMed
Tan LK, Giri D, Hummer AJ, Panageas KS, Brogi E, Norton L, Hudis C, Borgen PI, Cody HS: Occult axillary node metastases in breast cancer are prognostically significant: results in 368 node-negative patients with 20-year follow-up. J Clin Oncol. 2008, 26: 1803-1809. 10.1200/JCO.2007.12.6425. CrossRefPubMed
Querzoli P, Pedriali M, Rinaldi R, Lombardi AR, Biganzoli E, Boracchi P, Ferretti S, Frasson C, Zanella C, Ghisellini S, Ambrogi F, Antolini L, Piantelli M, Iacobelli S, Marubini E, Alberti S, Nenci I: Axillary lymph node nanometastases are prognostic factors for disease-free survival and metastatic relapse in breast cancer patients. Clin Cancer Res. 2006, 12: 6696-6701. 10.1158/1078-0432.CCR-06-0569. CrossRefPubMed
Brunsvig PF, Flatmark K, Aamdal S, Hoifodt H, Le H, Jakobsen E, Sandstad B, Fodstad O: Bone marrow micrometastases in advanced stage non-small cell lung carcinoma patients. Lung Cancer (Amsterdam, Netherlands). 2008, 61: 170-176. CrossRef
Hardingham JE, Kotasek D, Farmer B, Butler RN, Mi JX, Sage RE, Dobrovic A: Immunobead-PCR: a technique for the detection of circulating tumor cells using immunomagnetic beads and the polymerase chain reaction. Cancer Res. 1993, 53: 3455-3458. PubMed
Eaton MC, Hardingham JE, Kotasek D, Dobrovic A: Immunobead RT-PCR: a sensitive method for detection of circulating tumor cells. Biotechniques. 1997, 22: 100-105. PubMed
Norwegian breast cancer group. [ http://nbcg.no/]
Delahaye M, van der Ham F, van der Kwast TH: Complementary value of five carcinoma markers for the diagnosis of malignant mesothelioma, adenocarcinoma metastasis, and reactive mesothelium in serous effusions. Diagn Cytopathol. 1997, 17: 115-120. 10.1002/(SICI)1097-0339(199708)17:2<115::AID-DC6>3.0.CO;2-F. CrossRefPubMed
Flatmark K, Bjornland K, Johannessen HO, Hegstad E, Rosales R, Harklau L, Solhaug JH, Faye RS, Soreide O, Fodstad O: Immunomagnetic detection of micrometastatic cells in bone marrow of colorectal cancer patients. Clin Cancer Res. 2002, 8: 444-449. PubMed
Universal ProbeLibrary Assay Design Center. [ https://www.roche-applied-science.com/sis/rtpcr/upl/index.jsp?id=uplct_030000]
Miksicek RJ, Myal Y, Watson PH, Walker C, Murphy LC, Leygue E: Identification of a novel breast- and salivary gland-specific, mucin-like gene strongly expressed in normal and tumor human mammary epithelium. Cancer Res. 2002, 62: 2736-2740. PubMed
Putz E, Witter K, Offner S, Stosiek P, Zippelius A, Johnson J, Zahn R, Riethmuller G, Pantel K: Phenotypic characteristics of cell lines derived from disseminated cancer cells in bone marrow of patients with solid epithelial tumors: establishment of working models for human micrometastases. Cancer Res. 1999, 59: 241-248. PubMed
Mani SA, Guo W, Liao MJ, Eaton EN, Ayyanan A, Zhou AY, Brooks M, Reinhard F, Zhang CC, Shipitsin M, Campbell LL, Polyak K, Brisken C, Yang J, Weinberg RA: The epithelial-mesenchymal transition generates cells with properties of stem cells. Cell. 2008, 133: 704-715. 10.1016/j.cell.2008.03.027. CrossRefPubMedPubMedCentral
Tamaki Y, Akiyama F, Iwase T, Kaneko T, Tsuda H, Sato K, Ueda S, Mano M, Masuda N, Takeda M, Tsujimoto M, Yoshidome K, Inaji H, Nakajima H, Komoike Y, Kataoka TR, Nakamura S, Suzuki K, Tsugawa K, Wakasa K, Okino T, Kato Y, Noguchi S, Matsuura N: Molecular detection of lymph node metastases in breast cancer patients: results of a multicenter trial using the one-step nucleic acid amplification assay. Clin Cancer Res. 2009, 15: 2879-2884. 10.1158/1078-0432.CCR-08-1881. CrossRefPubMed
Martin Martinez MD, Veys I, Majjaj S, Lespagnard L, Schobbens JC, Rouas G, Filippov V, Noterman D, Hertens D, Feoli F, Bourgeois P, Durbecq V, Larsimont D, Nogaret JM: Clinical validation of a molecular assay for intra-operative detection of metastases in breast sentinel lymph nodes. Eur J Surg Oncol. 2009, 35: 387-392. 10.1016/j.ejso.2008.05.008. CrossRefPubMed
Viale G, Dell'Orto P, Biasi MO, Stufano V, De Brito Lima LN, Paganelli G, Maisonneuve P, Vargo JM, Green G, Cao W, Swijter A, Mazzarol G: Comparative evaluation of an extensive histopathologic examination and a real-time reverse-transcription-polymerase chain reaction assay for mammaglobin and cytokeratin 19 on axillary sentinel lymph nodes of breast carcinoma patients. Ann Surg. 2008, 247: 136-142. 10.1097/SLA.0b013e318157d22b. CrossRefPubMed
Effenberger KE, Borgen E, Eulenburg CZ, Bartkowiak K, Grosser A, Synnestvedt M, Kaaresen R, Brandt B, Nesland JM, Pantel K, Naume B: Detection and clinical relevance of early disseminated breast cancer cells depend on their cytokeratin expression pattern. Breast Cancer Res Treat. 2011, 125: 729-738. 10.1007/s10549-010-0911-2. CrossRefPubMed
Oncolex database. [ http://oncolex.no]
Pachmann K, Camara O, Kavallaris A, Krauspe S, Malarski N, Gajda M, Kroll T, Jorke C, Hammer U, Altendorf-Hofmann A, Rabenstein C, Pachmann U, Runnebaum I, Hoffken K: Monitoring the response of circulating epithelial tumor cells to adjuvant chemotherapy in breast cancer allows detection of patients at risk of early relapse. J Clin Oncol. 2008, 26: 1208-1215. 10.1200/JCO.2007.13.6523. CrossRefPubMed
Watson MA, Ylagan LR, Trinkaus KM, Gillanders WE, Naughton MJ, Weilbaecher KN, Fleming TP, Aft RL: Isolation and molecular profiling of bone marrow micrometastases identifies TWIST1 as a marker of early tumor relapse in breast cancer patients. Clin Cancer Res. 2007, 13: 5001-5009. 10.1158/1078-0432.CCR-07-0024. CrossRefPubMedPubMedCentral
Riethdorf S, Fritsche H, Muller V, Rau T, Schindlbeck C, Rack B, Janni W, Coith C, Beck K, Janicke F, Jackson S, Gornet T, Cristofanilli M, Pantel K: Detection of circulating tumor cells in peripheral blood of patients with metastatic breast cancer: a validation study of the CellSearch system. Clin Cancer Res. 2007, 13: 920-928. 10.1158/1078-0432.CCR-06-1695. CrossRefPubMed
Allard WJ, Matera J, Miller MC, Repollet M, Connelly MC, Rao C, Tibbe AG, Uhr JW, Terstappen LW: Tumor cells circulate in the peripheral blood of all major carcinomas but not in healthy subjects or patients with nonmalignant diseases. Clin Cancer Res. 2004, 10: 6897-6904. 10.1158/1078-0432.CCR-04-0378. CrossRefPubMed
Rao CG, Chianese D, Doyle GV, Miller MC, Russell T, Sanders RA, Terstappen LW: Expression of epithelial cell adhesion molecule in carcinoma cells present in blood and primary and metastatic tumors. Int J Oncol. 2005, 27: 49-57. PubMed
Raimondi C, Gradilone A, Naso G, Vincenzi B, Petracca A, Nicolazzo C, Palazzo A, Saltarelli R, Spremberg F, Cortesi E, Gazzaniga P: Epithelial-mesenchymal transition and stemness features in circulating tumor cells from breast cancer patients. Breast Cancer Res Treat. 2011.
Sorlie T, Perou CM, Tibshirani R, Aas T, Geisler S, Johnsen H, Hastie T, Eisen MB, van de Rijn M, Jeffrey SS, Thorsen T, Quist H, Matese JC, Brown PO, Botstein D, Eystein Lonning P, Borresen-Dale AL: Gene expression patterns of breast carcinomas distinguish tumor subclasses with clinical implications. Proc Natl Acad Sci USA. 2001, 98: 10869-10874. 10.1073/pnas.191367098. CrossRefPubMedPubMedCentral
Perou CM, Sorlie T, Eisen MB, van de Rijn M, Jeffrey SS, Rees CA, Pollack JR, Ross DT, Johnsen H, Akslen LA, Fluge O, Pergamenschikov A, Williams C, Zhu SX, Lonning PE, Borresen-Dale AL, Brown PO, Botstein D: Molecular portraits of human breast tumours. Nature. 2000, 406: 747-752. 10.1038/35021093. CrossRefPubMed
Park SY, Lee HE, Li H, Shipitsin M, Gelman R, Polyak K: Heterogeneity for stem cell-related markers according to tumor subtype and histologic stage in breast cancer. Clin Cancer Res. 16: 876-887.
- Analysis of EpCAM positive cells isolated from sentinel lymph nodes of breast cancer patients identifies subpopulations of cells with distinct transcription profiles
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