The online version of this article (doi:10.1186/bcr3622) contains supplementary material, which is available to authorized users.
Maryam B Lustberg, Priya Balasubramanian, Charles L Shapiro and Jeffrey J Chalmers contributed equally to this work.
The authors declare that they have no competing interests.
MBL, PB, CLS and JJC conceived of and designed the study. All authors collected and assembled the data. XP, MBL, PB, CLS and JJC analyzed and interpreted the data and wrote the manuscript. All authors read and approved the final manuscript.
Circulating tumor cells (CTCs) are commonly isolated from the blood by targeting the epithelial cell adhesion molecule (EpCAM) through positive selection. However, EpCAM can be downregulated during metastatic progression, or it can be initially not present. We designed the present prospective trial to characterize CTCs as well as other circulating cell populations in blood samples from women with metastatic breast cancer without EpCAM-dependent enrichment and/or isolation technology.
A total of 32 patients with metastatic breast cancer were enrolled, and blood samples were processed using a previously described negative depletion immunomagnetic methodology. Samples from healthy volunteers were run as controls (n = 5). Multistep sequential labeling was performed to label and fix cell-surface markers followed by permeabilization for cytokeratins (CK) 8, 18 and 19. Multiparametric flow cytometry (FCM) analysis was conducted using a BD LSR II flow cytometer or a BD FACSAria II or FACSAria III cell sorter. Immunocytochemical staining on postenrichment specimens for DAPI, EpCAM, CD45, CK, epidermal growth factor receptor and vimentin was performed. Expression of these markers was visualized using confocal microscopy (CM).
CD45-negative/CK-positive (CD45− CK+) populations with EpCAM + and EpCAM − expression were identified with both FCM and CM from the negatively enriched patient samples. In addition, EpCAM + and EpCAM − populations that were CK + and coexpressing the pan-hematopoietic marker CD45 were also noted. There were more CK + EpCAM − events/ml than CK + EpCAM + events/ml in both the CD45− and CD45+ fractions (both statistically significant at P ≤ 0.0005). The number of CK + CD45− and CK + CD45+ events per milliliter in blood samples (regardless of EpCAM status) was higher in patient samples than in normal control samples (P ≤ 0.0005 and P ≤ 0.026, respectively). Further, a significant fraction of the CK + CD45+ events also expressed CD68, a marker associated with tumor-associated macrophages. Higher levels of CD45-CK + EpCAM − were associated with worse overall survival (P = 0.0292).
Metastatic breast cancer patients have atypical cells that are CK + EpCAM − circulating in their blood. Because a substantial number of these patients do not have EpCAM + CTCs, additional studies are needed to evaluate the role of EpCAM − circulating cells as a prognostic and predictive marker.
Additional file 1: Table S1: Antibodies used. 4′,6-diamidino-2-phenylindole (DAPI). Alexa Fluor (AF). (DOC 38 KB)13058_2013_3445_MOESM1_ESM.doc
Additional file 2: Supplemental Information.(DOCX 693 KB)13058_2013_3445_MOESM2_ESM.docx
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Nagrath S, Sequist LV, Maheswaran S, Bell DW, Irimia D, Ulkus L, Smith MR, Kwak EL, Digumarthy S, Muzikansky A, Ryan P, Balis UJ, Tompkins RG, Haber DA, Toner M: Isolation of rare circulating tumour cells in cancer patients by microchip technology. Nature. 2007, 450: 1235-1239. 10.1038/nature06385. CrossRefPubMedPubMedCentral
Mayer JA, Pham T, Wong KL, Scoggin J, Sales EV, Clarin T, Pircher TJ, Mikolajczyk SD, Cotter PD, Bischoff FZ: FISH-based determination of HER2 status in circulating tumor cells isolated with the microfluidic CEE™ platform. Cancer Genet. 2011, 204: 589-595. 10.1016/j.cancergen.2011.10.011. CrossRefPubMed
Pachmann K, Dengler R, Lobodasch K, Fröhlich F, Kroll T, Rengsberger M, Schubert R, Pachmann U: An increase in cell number at completion of therapy may develop as an indicator of early relapse: quantification of circulating epithelial tumor cells (CETC) for monitoring of adjuvant therapy in breast cancer. J Cancer Res Clin Oncol. 2008, 134: 59-65. CrossRefPubMed
Institut Curie: CirCé01 Study: Evaluation of the use of circulating tumour cells to guide chemotherapy from the third line of chemotherapy for metastatic breast cancer. ClinicalTrials.gov Identifier: NCT01349842. http://clinicaltrials.gov/show/NCT01349842
Riethdorf S, Fritsche H, Müller V, Rau T, Schindlbeck C, Rack B, Janni W, Coith C, Beck K, Jänicke 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 Cell Search system. Clin Cancer Res. 2007, 13: 920-928. 10.1158/1078-0432.CCR-06-1695. CrossRefPubMed
Balasubramanian P, Lang JC, Jatana KR, Miller B, Ozer E, Old M, Schuller DE, Agrawal A, Teknos TN, Summers TA, Lustberg MB, Zborowski M, Chalmers JJ: Multiparameter analysis, including EMT markers, on negatively enriched blood samples from patients with squamous cell carcinoma of the head and neck. PLoS One. 2012, 7: e42048-10.1371/journal.pone.0042048. CrossRefPubMedPubMedCentral
Jatana KR, Balasubramanian P, Lang JC, Yang L, Jatana CA, White E, Agrawal A, Ozer E, Schuller DE, Teknos TN, Chalmers JJ: Significance of circulating tumor cells in patients with squamous cell carcinoma of the head and neck: initial results. Arch Otolaryngol Head Neck Surg. 2010, 136: 1274-1279. 10.1001/archoto.2010.223. CrossRefPubMedPubMedCentral
Lustberg M, Jatana KR, Zborowski M, Chalmers JJ: Emerging technologies for CTC detection based on depletion of normal cells. Recent Res Cancer Res. 2012, 195: 97-110. 10.1007/978-3-642-28160-0_9. CrossRef
Yu M, Bardia A, Wittner BS, Stott SL, Smas ME, Ting DT, Isakoff SJ, Ciciliano JC, Wells MN, Shah AM, Concannon KF, Donaldson MC, Sequist LV, Brachtel E, Sgroi D, Baselga J, Ramaswamy S, Toner M, Haber DA, Maheswaran S: Circulating breast tumor cells exhibit dynamic changes in epithelial and mesenchymal composition. Science. 2013, 339: 580-584. 10.1126/science.1228522. CrossRefPubMedPubMedCentral
Powell AA, Talasaz AH, Zhang H, Coram MA, Reddy A, Deng G, Telli ML, Advani RH, Carlson RW, Mollick JA, Sheth S, Kurian AW, Ford JM, Stockdale FE, Quake SR, Pease RF, Mindrinos MN, Bhanot G, Dairkee SH, Davis RW, Jeffrey SS: Single cell profiling of circulating tumor cells: transcriptional heterogeneity and diversity from breast cancer cell lines. PLoS One. 2012, 7: e33788-10.1371/journal.pone.0033788. CrossRefPubMedPubMedCentral
Yang L, Lang JC, Balasubramanian P, Jatana KR, Schuller D, Agrawal A, Zborowski M, Chalmers JJ: Optimization of an enrichment process for circulating tumor cells from the blood of head and neck cancer patients through depletion of normal cells. Biotechnol Bioeng. 2009, 102: 521-534. 10.1002/bit.22066. CrossRefPubMedPubMedCentral
Tong X, Yang L, Lang JC, Zborowski M, Chalmers JJ: Application of immunomagnetic cell enrichment in combination with RT-PCR for the detection of rare circulating head and neck tumor cells in human peripheral blood. Cytometry B Clin Cytom. 2007, 72B: 310-323. 10.1002/cyto.b.20177. CrossRef
Zimmerlin L, Donnenberg VS, Donnenberg AD: Rare event detection and analysis in flow cytometry: bone marrow mesenchymal stem cells, breast cancer stem/progenitor cells in malignant effusions, and pericytes in disaggregated adipose tissue. Methods Mol Biol. 2011, 699: 251-273. 10.1007/978-1-61737-950-5_12. CrossRefPubMed
Stott SL, Hsu CH, Tsukrov DI, Yu M, Miyamoto DT, Waltman BA, Rothenberg SM, Shah AM, Smas ME, Korir GK, Floyd FP, Gilman AJ, Lord JB, Winokur D, Springer S, Irimia D, Nagrath S, Sequist LV, Lee RJ, Isselbacher KJ, Maheswaran S, Haber DA, Toner M: Isolation of circulating tumor cells using a microvortex-generating herringbone-chip. Proc Natl Acad Sci USA. 2010, 107: 18392-18397. 10.1073/pnas.1012539107. CrossRefPubMedPubMedCentral
Budd GT, Cristofanilli M, Ellis MJ, Stopeck A, Matera J, Miller MC, Doyle GV, Allard WJ, Terstappen LW, Hayes DF: Monitoring circulating tumor cells (CTC) in non-measurable metastatic breast cancer (MBC). J Clin Oncol. 2005, 23 (16 Suppl): 503-
Budd GT, Cristofanilli M, Ellis MJ, Stopeck A, Borden E, Miller MC, Matera J, Repollet M, Doyle GV, Terstappen LWWM, Hayes DF: Circulating tumor cells versus imaging—predicting overall survival in metastatic breast cancer. Clin Cancer Res. 2006, 12: 6403-6409. 10.1158/1078-0432.CCR-05-1769. CrossRefPubMed
Hayes DF, Cristofanilli M, Budd GT, Ellis MJ, Stopeck A, Miller MC, Matera J, Allard WJ, Doyle GV, Terstappen LWWM: Circulating tumor cells at each follow-up time point during therapy of metastatic breast cancer patients predict progression-free and overall survival. Clin Cancer Res. 2006, 12: 4218-4224. 10.1158/1078-0432.CCR-05-2821. CrossRefPubMed
Liu MC, Shields PG, Warren RD, Cohen P, Wilkinson M, Ottaviano YL, Rao SB, Eng-Wong J, Seillier-Moiseiwitsch F, Noone AM, Isaacs C: Circulating tumor cells: a useful predictor of treatment efficacy in metastatic breast cancer. J Clin Oncol. 2009, 27: 5153-5159. 10.1200/JCO.2008.20.6664. CrossRefPubMedPubMedCentral
Sieuwerts AM, Kraan J, Bolt J, van der Spoel P, Elstrodt F, Schutte M, Martens JW, Gratama JW, Sleijfer S, Foekens JA: Anti-epithelial cell adhesion molecule antibodies and the detection of circulating normal-like breast tumor cells. J Natl Cancer Inst. 2009, 101: 61-66. 10.1093/jnci/djn419. CrossRefPubMedPubMedCentral
Müller V, Riethdorf S, Rack B, Janni W, Fasching PA, Solomayer E, Aktas B, Kasimir-Bauer S, Pantel K, Fehm T, on behalf of the DETECT study group: Prognostic impact of circulating tumor cells assessed with the Cell Search System™ and AdnaTest Breast™ in metastatic breast cancer patients: the DETECT study. Breast Cancer Res. 2012, 14: R118-10.1186/bcr3243. CrossRefPubMedPubMedCentral
Wallwiener M, Hartkopf AD, Baccelli I, Riethdorf S, Schott S, Pantel K, Marmé F, Sohn C, Trumpp A, Rack B, Aktas B, Solomayer EF, Müller V, Janni W, Schneeweiss A, Fehm TN: The prognostic impact of circulating tumor cells in subtypes of metastatic breast cancer. Breast Cancer Res Treat. 2013, 137: 503-510. 10.1007/s10549-012-2382-0. CrossRefPubMed
Giordano A, Giuliano M, De Laurentiis M, Arpino G, Jackson S, Handy BC, Ueno NT, Andreopoulou E, Alvarez RH, Valero V, De Placido S, Hortobagyi GN, Reuben JM, Cristofanilli M: Circulating tumor cells in immunohistochemical subtypes of metastatic breast cancer: lack of prediction in HER2-positive disease treated with targeted therapy. Ann Oncol. 2012, 23: 1144-1150. 10.1093/annonc/mdr434. CrossRefPubMed
Jiang ZF, Cristofanilli M, Shao ZM, Tong ZS, Song EW, Wang XJ, Liao N, Hu XC, Liu Y, Wang Y, Zeng L, Zhang M: Circulating tumor cells predict progression-free and overall survival in Chinese patients with metastatic breast cancer, HER2-positive or triple-negative (CBCSG004): a multicenter, double-blind, prospective trial. Ann Oncol. 2013, 24: 2766-2772. 10.1093/annonc/mdt246. CrossRefPubMed
Zhang L, Ridgway LD, Wetzel MD, Ngo J, Yin W, Kumar D, Goodman JC, Groves MD, Marchetti D: The identification and characterization of breast cancer CTCs competent for brain metastasis. Sci Transl Med. 2013, 5: 180ra148-A published erratum appears in Sci Transl Med 2013, 5:189er5 CrossRef
DeNardo DG, Brennan DJ, Rexhepaj E, Ruffell B, Shiao SL, Madden SF, Gallagher WM, Wadhwani N, Keil SD, Junaid SA, Rugo HS, Hwang ES, Jirström K, West BL, Coussens LM: Leukocyte complexity predicts breast cancer survival and functionally regulates response to chemotherapy. Cancer Discov. 2011, 1: 54-67. 10.1158/2159-8274.CD-10-0028. CrossRefPubMedPubMedCentral
- Heterogeneous atypical cell populations are present in blood of metastatic breast cancer patients
Maryam B Lustberg
Erin R Macrae
Rachel M Layman
Thomas A Summers
Charles L Shapiro
Jeffrey J Chalmers
- BioMed Central
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