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Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like Breast Tumor Cells

Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like... BRIEF COMMUNICATION preparation system), the CellSearch epithe- Anti-Epithelial Cell Adhesion Molecule lial cell kit (to enrich for cells expressing Antibodies and the Detection of EpCAM and to label nuclei, leukocytes, and epithelial cells), and the CellSpotter Circulating Normal-Like Breast Tumor Analyzer (a semiautomated fl uorescence- based microscopy system that permits Cells computer-generated reconstruction of cel- lular images). By use of the CellSearch Anieta M. Sieuwerts , Jaco Kraan , Joan Bolt , Petra van der Spoel , Fons Elstrodt , epithelial cell kit, circulating tumor cells Mieke Schutte , John W. M. Martens , Jan-Willem Gratama , Stefan Sleijfer , John are isolated with anti-EpCAM antibodies A. Foekens coupled to microscopic iron particles, and Identification of specific subtypes of circulating tumor cells in peripheral blood of complexes of circulating tumor cells bound cancer patients can provide information about the biology of metastasis and to anti-EpCAM – coupled iron particles are improve patient management. However, to be effective, the method used to iden- “pulled” out of the blood sample by use of tify circulating tumor cells must detect all tumor cell types. We investigated whether powerful magnets. Unbound cells and the the five subtypes of human breast cancer cells that have been defined by global remaining plasma are aspirated, and the gene expression profiling — normal-like, basal, HER2-positive, and luminal A and immunomagnetically isolated cells are per- B — were identified by CellSearch, a US Food and Drug Administration – approved meabilized and stained with 4 ′ ,6-diamidino- test that uses antibodies against the cell surface – expressed epithelial cell adhesion 2-phenylindole (to detect nuclei); molecule (EpCAM) to isolate circulating tumor cells. We used global gene expres- anti-CD45 antibodies labeled with allophy- sion profiling to determine the subtypes of a well-defined panel of 34 human breast cocyan (to detect leukocytes); and anti- cancer cell lines (15 luminal, nine normal-like, five basal-like, and five Her2-positive). cytokeratin 8, 18, and 19 antibodies labeled We mixed 50-150 cells from 10 of these cell lines with 7.5 mL of blood from a single with phycoerythrin (to detect epithelial healthy human donor, and the mixtures were subjected to the CellSearch test to cells). After incubation with staining isolate the breast cancer cells. We found that the CellSearch isolation method, reagents, the magnetic separation is which uses EpCAM on the surface of circulating tumor cells for cell isolation, did repeated and excess staining reagents are not recognize, in particular, normal-like breast cancer cells, which in general have removed by aspiration. In the fi nal process- aggressive features. New tests that include antibodies that specifically recognize ing step, the immunomagnetically isolated normal-like breast tumor cells but not cells of hematopoietic origin are needed. cells are resuspended in a MagNest Cell Presentation Device (Veridex LLC). This J Natl Cancer Inst 2009;101: 61 – 66 device consists of a chamber and two mag- nets that orient the immunomagnetically Circulating tumor cells are cells that have However, it is unclear whether such tests labeled cells for analysis by use of the detached from the primary tumor or meta- can detect all tumor subtypes. We investi- static tumor sites and entered the periph- gated whether the CellSearch test could eral circulation. A limited number of recognize all subtypes of breast cancer, Affiliations of authors: Department of Medical markers are used for the isolation (ie, cell including normal-like, basal, HER2- Oncology, Josephine Nefkens Institute and Cancer surface antigens) or detection (ie, various positive, and luminal A and B tumor cells. Genomics Centre (AMS, JB, JWMM, JAF), Department of Medical Oncology, Daniel den antigens or mRNAs) of circulating tumor The CellSearch circulating tumor cell Hoed Cancer Center (JK, PvdS, J-WG, SS), and cells. These markers include epithelial cell test is the only diagnostic test that is cur- Department of Medical Oncology, Josephine surface markers, such as the epithelial cell rently approved by the US Food and Drug Nefkens Institute (FE, MS), Erasmus MC, Rotterdam, the Netherlands . adhesion molecule (EpCAM; also known as Administration as an automated test to Correspondence to: Anieta M. Sieuwerts, PhD, CD326, ESA, HEA125, and TACSTD1); detect and enumerate circulating tumor Department of Medical Oncology, Josephine cytokeratins 7, 8, 18, 19, and 20; and more cells ( 4 ). Briefl y, a blood sample that con- Nefkens Institute, Erasmus MC, Rm BE-400, Dr cancer-specific markers, such as HER2- tains many leukocytes and few circulating Molewaterplein 50, 3015 GE Rotterdam, the Netherlands (e-mail: a.sieuwerts@erasmusmc.nl ). neu and mucin 1 for breast carcinoma tumor cells is drawn into 10-mL CellSave See “Funding” and “Notes” following “References.” ( 1 – 3 ). Commercially available tests for iso- Preservative Tubes (Veridex LCC), which DOI: 10.1093/jnci/djn419 lation and detection of circulating tumor contain EDTA as an anticoagulant and a © 2008 The Author(s). cells include the CellSearch circulating cellular preservative. The blood is main- This is an Open Access article distributed under tumor cell test (Veridex LLC, San Diego, tained at room temperature and subse- the terms of the Creative Commons Attribution CA) and other tests ( 1 – 3 ). These tests use quently processed within 72 hours of Non-Commercial License (http://creativecommons. org/licenses/by-nc/2.0/uk/), which permits unre- combinations of specific antibodies against collection by use of the CellSearch system stricted non-commercial use, distribution, and these molecules and generally include anti- (Veridex LLC), which consists of the reproduction in any medium, provided the origi- bodies against EpCAM for cell isolation. CellTracks Autoprep (an automated sample nal work is properly cited. jnci.oxfordjournals.org JNCI | Brief Communication 61 patients who have responded to systemic chain reaction. The 14 candidate genes CONTEXT AND CAVEATS therapy (who have a decreased number of that may as such enable dis crimination Prior knowledge circulating tumor cells) from those who between the breast cancer subgroups Identification of specific subtypes of circu- have not (who have an increased number of included genes that are more specifi c for lating tumor cells in the peripheral blood of circulating tumor cells) ( 15 ). cells of hematopoietic origin ( CD44 and cancer patients can provide important prog- Five subtypes of human breast cancers CD45 ); epithelial cell – specifi c genes such nostic information, but to be effective, the have been identifi ed by global gene expres- as those encoding cytokeratins, EpCAM, method used must recognize all tumor cell sion profi ling, including normal-like, basal, and mucin 1; genes encoding markers types. HER2-positive, and luminal A and B ( 16 ). specifi c for the breast cancer subgroups Study design The normal-like and basal subtypes, which ( ESR1 , ERBB1 , ERBB2 , CAV1 , and CD24 ); The subtype of 19 well-characterized breast represented 7.8% and 25% of all breast and genes for two well-known epithelial – cancer cell lines was obtained by use of cancers, respectively, in a cohort of 344 mesenchymal transition markers ( TWIST1 gene expression profiling, including normal- breast tumor samples from patients with and VIM ). Transcript levels of these 14 like, basal-like, HER2-positive, and luminal lymph node – negative disease ( 17 ), are in genes in all the 34 cell lines were compared A and B. Cells from each line were mixed general negative for estrogen receptor with those measured in whole-blood cells with blood from a healthy donor and sub- ( ESR1 ), progesterone receptor, and HER2- from 23 different blood donors before (n = jected to the CellSearch circulating tumor neu, and so lack the molecular targets for 6 samples) and after (n = 23 samples) being cell assay. endocrine therapy and anti-HER2 – directed subjected to EpCAM-based CellSearch Contribution therapy and therefore have worse treat- enrichment to establish which genes were The CellSearch assay, which uses epithelial ment options than other subtypes. Because not expressed by blood cells and could be cell adhesion molecules on the cell surface, each breast cancer subtype has distinct used to specifi cally identify the epithelial did not recognize normal-like breast can - prognostic and therapeutic characteristics tumor cells ( Table 1 ). The study was cer cells, although other subtypes were ( 16 , 18 ), it is important to establish whether approved by the Erasmus MC Institutional recognized. each breast cancer subtype expresses the Review Board. Blood samples were col- Implications cell surface antigens that are used in assays lected from healthy volunteers after written Normal-like breast cancer cells have espe- to isolate and detect circulating tumor informed consent was obtained. Supple- cially aggressive features, and so assays cells. It is also important to identify which mentary Table 1 (available online) lists the that recognize this subtype would provide breast cancer subtypes do not express an gene expression assays used. valuable prognostic information. New antigen and to determine which antibodies We next randomly selected 19 of the 34 assays are needed that include antibodies could be used instead of or in addition to cell lines; incubated each cell line with that specifically recognize this breast can- cer subtype but not other cell types, includ- antibodies that are included in the test for fl uorochrome-conjugated antibodies against ing those of hematopoietic origin. circulating tumor cells. CD45, CD24, CD44, or EpCAM, which To investigate whether the CellSearch were chosen to enable a distinction between Limitations test recognized all fi ve breast cancer sub- CD45-positive cells of hematopoietic ori- Only homogeneous breast cancer cell lines types that are characterized by their intrin- gin, CD45-negative and EpCAM-positive with known subtypes were investigated. sic gene expression profi les as previously breast cancer cells, and CD45-negative, From the Editors described ( 16 , 18 ), we fi rst determined the CD24-negative, and CD44-positive breast subtypes of our well-defi ned panel of 34 cancer stem cells ( 21 ); and used fl ow cytom- CellSpotter Analyzer, a four-color semiau- human breast cancer cell lines ( 19 ) by etry to measure the expression of each tomated fl uorescence microscope. Finally, global gene expression profi ling, as marker by each cell line ( Table 2 ). Next, an circulating tumor cells that are defi ned as described previously ( 17 ). A previous analy- aliquot (50 – 150 cells per aliquot) of each of nucleated cells with a round to oval mor- sis of nearly 150 polymorphic microsatellite these 19 cell lines was added to a separate phology that are positive for anti- markers in this panel of 34 cell lines has 7.5-mL sample of peripheral blood from a cytokeratin antibody binding and negative shown that each of these 34 cell lines is single healthy volunteer donor, and the for anti-CD45 antibody binding ( 5 ) are unique and monoclonal ( 20 ). As determined mixtures were subjected to the EpCAM- identifi ed by an operator. by the expression of genes in the intrinsic based CellSearch assay to isolate circulat - Results of the CellSearch test have been gene set ( 16 , 17 ), the complete panel con- ing tumor cells. Gene expression data of used to monitor disease progression and tained 15 luminal cell lines (ie, no clear-cut the isolated cells showed that cells with a therapy effi cacy in metastatic prostate ( 6 ), discrimination between luminal A and B), luminal or HER2-positive subtype were colorectal ( 7 ), and breast ( 8 ) cancer. For nine normal-like lines, fi ve basal-like lines, generally isolated by the assay, whereas patients with advanced breast cancer, the and fi ve HER2-positive cell lines ( Table 1 ). cells with a normal-like subtype, which lack number of circulating tumor cells as deter- To validate the gene expression profi ling EpCAM expression, were not isolated or mined by the CellSearch test has been data, transcript levels of 14 candidate genes were only partially isolated (eg, MDA-MB- shown to have prognostic value ( 8 – 14 ). In were remeasured with Affymetrix GeneChip 231 cells, which have marginal EpCAM addition, a change from the baseline num- Exon 1.0 ST Arrays (Affymetrix UK Ltd., expression) ( Figure 1 ; Supplementary ber of circulating tumor cells after the fi rst Wickham la Wooburn Grn, UK) and Table 2 , available online). We found that cycle of therapy appears to distinguish real-time reverse transcriptase – polymerase the cell lines with the normal-like subtype 62 Brief Communication | JNCI Vol. 101, Issue 1 | January 7, 2009 jnci.oxfordjournals.org JNCI | Brief Communication 63 Table 1 . mRNA expression in breast cancer cell lines with different intrinsic subtype characteristics * Relative mRNA expression (95% CI) Relative mRNA expression (95% CI) Blood before Blood after Normal-like Basal-like Luminal HER2-positive enrichment enrichment Gene symbol (antigen) (n = 9) (n = 5) (n = 15) (n = 5) P † (n = 6) (n = 23) PTPRC (CD45) 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) .210 13.39 (10.42 to 16.37) 6.25 (4.99 to 7.50) CD24 (CD24) 0.40 (0.03 to 0.78) 2.31 (0.20 to 4.42) 3.84 (2.14 to 5.54) 2.69 (1.86 to 3.52) .025 0.03 (0.02 to 0.03) 0.03 (0.01 to 0.06) CD44 (CD44) 0.77 (0.59 to 0.95) 0.75 (0.40 to 1.10) 0.04 (0.02 to 0.06) 0.02 (0.00 to 0.04) <.001 3.85 (3.13 to 4.57) 3.90 (3.29 to 4.52) TACSTD1 (EpCAM) 0.04 (0.00 to 0.10) 2.04 (0.83 to 3.25) 8.72 (4.02 to 13.43) 4.43 (2.17 to 6.69) .020 0.00 0.01 (0.00 to 0.02) MUC1 (EMA, mucin 1) 0.32 (0.20 to 0.44) 0.35 (0.15 to 0.55) 1.88 (1.24 to 2.52) 0.53 (0.43 to 0.63) <.001 0.03 (0.02 to 0.05) 0.01 (0.00 to 0.02) CAV1 (caveolin 1) 5.04 (2.39 to 7.70) 1.70 (0.19 to 3.21) 0.05 (0.02 to 0.08) 0.03 (0.01 to 0.05) <.001 0.02 (0.01 to 0.04) 0.05 (0.00 to 0.01) KRT7 (cytokeratin 7) 1.53 (0.00 to 3.07) 7.90 (5.63 to 10.17) 6.62 (3.62 to 9.63) 10.46 (6.45 to 14.48) .009 0.01 (0.00 to 0.02) 0.01 (0.00 to 0.01) KRT18 (cytokeratin 18) 5.58 (0.96 to 10.19) 10.06 (7.88 to 12.24) 16.80 (13.23 to 20.36) 17.17 (12.11 to 22.23) .001 0.06 (0.03 to 0.09) 0.03 (0.02 to 0.03) KRT19 (cytokeratin 19) 0.09 (0.00 to 0.26) 4.53 (0.00 to 9.11) 28.35 (12.66 to 44.03) 11.99 (1.58 to 22.40) .022 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) ESR1 (estrogen receptor) 0.00 (0.00 to 0.00) 0.02 (0.01 to 0.03) 0.76 (0.37 to 1.14) 0.06 (0.00 to 0.16) .003 0.02 (0.01 to 0.03) 0.01 (0.00 to 0.02) ERBB1 (EGF receptor) 0.20 (0.10 to 0.30) 0.96 (0.00 to 2.01) 0.03 (0.01 to 0.05) 0.07 (0.00 to 0.14) .003 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) ERBB2 (HER2) 0.05 (0.03 to 0.06) 0.07 (0.02 to 0.12) 2.53 (0.13 to 4.94) 7.69 (2.28 to 13.09) .009 0.02 (0.01 to 0.03) 0.01 (0.00 to 0.01) TWIST1 (EMT marker TWIST1) 0.06 (0.01 to 0.11) 0.00 (0.00 to 0.00) 0.01 (0.00 to 0.02) 0.00 (0.00 to 0.00) .027 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) VIM (EMT marker vimentin) 101.56 (36.90 to 116.23) 0.43 (0.00 to 0.90) 0.30 (0.00 to 0.67) 0.10 (0.00 to 0.27) <.001 34.47 (22.12 to 46.82) 52.30 (44.10 to 60.50) * Data in this table are the average and its 95% CI of one representative experiment. Results from other experiments were similar. Expression data are presented relative to that of HMBS , HPRT1 , and GUSB. Fourteen candidate genes were selected, including genes that are more specific for cells of hematopoietic origin ( CD44 and CD45 ); epithelial cell – specific genes such as those encoding cytokeratins, EpCAM, and mucin 1; genes encoding markers specific for the breast cancer subgroups ( ESR1 , ERBB1 , ERBB2 , CAV1 , and CD24 ); and genes for two well-known EMT markers ( TWIST1 and VIM ). Data of the individual cell lines are presented in Supplementary Table 3 (available online), and the gene expression assays used are shown in Supplementary Table 1 (available online). To validate the gene expression profiling data, transcript levels of candidate genes that might enable discrimination among the four breast cancer subtypes, as represented by 19 randomly selected breast cancer cell lines, were analyzed with Affymetrix GeneChip Exon 1.0 ST Arrays (Affymetrix UK Ltd., Wickham la Wooburn Grn, UK) and real-time RT-PCR and compared with levels measured in whole blood of healthy blood donors before (n = 6) or after (n = 23) EpCAM-based CellSearch enrichment for circulating tumor cells. RNA was isolated from healthy blood and cell lines with the RNeasy (Micro) kit (Qiagen BV, Venlo, the Netherlands). For the cell lines, cDNA was prepared by use of the Superscript II RNase H-kit from Invitrogen (Breda, the Netherlands). For healthy blood donors before CellSearch enrichment and the preparations from healthy blood donors and cell lines after CellSearch enrichment, cDNA was synthesized with the RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, St Leon-Rot, Germany), followed by a linear preamplification for the non-[2-[ N -(3-dimethylaminopropyl)- N -propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium] (SYBR Green I)-based assays (TaqMan PreAmp from Applied Biosystems, Nieuwerkerk a/d IJssel, the Netherlands), according to the manufacturer ’ s instructions. The resulting cDNA preparations were analyzed by real-time PCR with TaqMan gene expression assays and TaqMan Universal PCR Master Mix No AmpErase UNG (Applied Biosystems) and Absolute qPCR SYBR Green I mixture (Abgene, Epsom, UK) for the SYBR-based assays. PCRs were performed in a 20- µ L reaction volume in an Mx3000P Real-Time PCR system (Stratagene, Amsterdam, the Netherlands). Expression of HMBS , HPRT1 , and GUSB was used as a reference to control sample loading and RNA quality, as described previously (26). To enable comparison between the different methods of measuring gene expression (Affymetrix GeneChip Exon 1.0 ST arrays and real-time RT-PCR), data were normalized by expression levels that were measured by both methods in a set of seven control cell lines (ie, MDA-MB-435s, MDA-MB-231, CAMA-1, MCF-7, T47D, SK-BR-3, and EVSA-T). EGF = epidermal growth factor receptor; EMA = epithelial membrane antigen; EpCAM = epithelial cell adhesion molecule; EMT = epithelial – mesenchymal transition; CI = confidence interval; RT-PCR = reverse transcriptase – polymerase chain reaction; cDNA = complementary DNA. † One-way analysis of variance was used to test for differences between the four subgroups present in the cell lines, with P < .05 being considered statistically significant. All statistical tests were two-sided. expressed high mRNA levels of the epithe- to enter the circulation and seed metastases vimentin and CD44 mRNA and the low lial – mesenchymal transition markers, ( 23 – 25 ). Thus, the normal-like breast can- expression of CD24 mRNAs. Because anti- TWIST1 ( TWIST1 ) and vimentin ( VIM ) cer cell subtype is an important target for bodies against vimentin and CD44 also (one-way analysis of variance, two-sided the development of individualized therapy bind to cells of hematopoietic origin, these P = .03 and P < .001, respectively) ( Table 1 ); and should not be overlooked when assess- antibodies would not be suitable for the however, MDA-MB-231 cells expressed ing circulating tumor cells. Intrinsic breast detection of circulating tumor cells that vimentin but did not appear to express cancer subtypes are easily missed in analy- have an epithelial – mesenchymal transition TWIST1 ( Supplementary Table 3 , avail- ses that use only crude, standard clinical or origin or putative breast cancer stem cells. able online). pathological criteria such as hormone However, current strategies for detecting Cells with a normal-like breast cancer receptor and HER2 status. Therefore, our circulating tumor cells can be improved to subtype express high levels of genes that results, which are based on a distinction in allow assessment of these normal-like cells are characteristic of basal epithelial and breast cancer subtypes determined by by selecting antigens that are ubiquitously adipose cells and low levels of genes that global gene expression profi ling, cannot be and abundantly present and that can be are characteristic of luminal epithelial cells compared with those of Cristofanilli et al. made accessible for isolation with immuno- ( 16 ). The normal-like breast cancer subtype ( 8 ), who did not fi nd differences in the beads on normal-like cells but that are is the only subtype that displays the puta- number of circulating tumor cells between absent on cells of hematopoietic origin. tive tumor-initiating stem cell phenotype, breast cancer cell subtypes that were defi ned When selecting antigens to use for isolat- which includes low expression of CD24 only by hormone receptor and HER2 ing circulating normal-like breast cancer and high expression of CD44 ( 21 , 22 ) status. cells, it must be emphasized that clinical ( Table 2 ) and has high expression of New tests that include antibodies that (breast) cancer samples consist of heteroge- both vimentin and TWIST1 ( Table 1 ). specifi cally recognize normal-like breast neous cell populations and always contain Expression of vimentin and TWIST1 has tumor cells but not cells of hematopoietic leukocytes and other types of blood cells; been used to identify cells that have under- origin are needed. As shown in Table 1 , the we circumvented this problem by using gone the epithelial – mesenchymal transi- phenotype of circulating leukocytes in the homogeneous cell lines. Examples of mem- tion, a process that has been linked to the healthy blood donors before and after brane antigens that might fulfi ll the criteria generation of cells with properties of stem EpCAM-based CellSearch enrichment was of being ubiquitously and abundantly pres- cells and to the ability of breast cancer cells characterized by the high expression of ent on normal-like cells but absent on cells Table 2 . Immunological assessment of antigens in breast cancer cell lines with different intrinsic subtype characteristics and circulating tumor cell recovery * Flow cytometry, † MFI CellSearch, ‡ % of cells Intrinsic subtype No. of cell lines CD45 CD24 CD44 EpCAM recovered (95% CI) Normal-like 6 <5 <5 >1000 <5 2 (0 to 6) § Basal-like 5 <5 5– 20 200 – 1000 20 – 200 48 (36 to 61) Luminal 5 <5 5– 20 5 – 20 20 – 200 75 (62 to 89) HER2-positive 3 <5 5– 20 <5 20 – 200 85 (61 to 108) * MFI = mean fluorescence intensity; EpCAM = epithelial cell adhesion molecule; CI = confidence interval; 7AAD = 7-amino-actinomycin D; FITC = fluorescein isothiocyanate. † Cultured human breast cancer cell lines were incubated with the following fluorochrome-conjugated monoclonal antibodies: EpCAM conjugated with FITC (clone EBA-1; BD Biosciences, San Jose, CA), CD24 conjugated with FITC (clone SN3; eBioscience, Inc., San Diego, CA), CD44 conjugated with R-phycoerythrin coupled to the cyanine dye Cy7 (PE-Cy7, clone HIB19; eBioscience), and CD45 conjugated with allophycocyanin (clone 2D1; BD Biosciences). We used 7AAD (Sigma-Aldrich, St Louis, MO; 1 µ g/mL) to control for viability. Cells were then analyzed on a Canto flow cytometer (BD Biosciences). To exclude nonviable cells from analysis, only 7AAD-negative (viable) cells were evaluated for antigen expression. Unstained cells were used as a negative control. Only cells that expressed EpCAM were captured by the anti-EpCAM antibody present in the CellSearch circulating tumor cell profile kit. In addition, as shown in Figure 1 and Supplementary Table 2 (available online), MDA-MB-231 cells with marginal EpCAM expression were only partially (10 of 82 added tumor cells, or approximately 12%) captured by the CellSearch antibody, indicating that anti-EpCAM antibodies from the EBA-1 clone (BD Biosciences) and from the CellSearch circulating tumor cell kit have similar sensitivities and specificities. ‡ Data are the average (95% CI) of one representative experiment performed in duplicate for the given number of cell lines. Blood samples containing EDTA (7.5-mL aliquots of blood) from a single healthy blood donor were obtained from CellSave Preservative Tubes (Veridex LCC, San Diego, CA). To each sample, 20 µ L of a cell suspension containing 50 – 150 cultured cells from the indicated subtype of human breast cancer was added. To determine the actual viable cell number, a 100- µ L aliquot of the cultured cells was incubated with 10 µ L of 7AAD (1 µ g/mL) and 100 µ L of fluorescent beads (Beckman – Coulter, Inc., Miami, FL). After a 15-minute incubation at room temperature, 2 mL of phosphate-buffered saline was added, and samples were analyzed on a Calibur flow cytometer (BD Biosciences). At least 10 000 beads were acquired to estimate the number of 7AAD-negative (viable) cells. The efficiency of retrieving the tumor cells was controlled by counting the exact number of viable cells that were drawn in triplicate by flow cytometry and by light microscopy after serial dilution. To establish the number of circulating tumor cells recovered, samples were processed on the CellTrack AutoPrep analyzer (Veridex LLC) with the CellSearch circulating tumor cell profile kit (Veridex LLC), which uses microscopic iron particles coupled to anti-EpCAM to enrich for circulating tumor cells. The number of circulating tumor cells (ie, cells stained with the nuclear dye, 4 ′ ,6-diamidino-2-phenylindole, that are positive for cytokeratin 8, 18, and 19, and negative for CD45) were determined on the CellSpotter analyzer (Veridex LLC), according to the manufacturer’s instructions (for full flow cytometry and CellSearch recovery data on these 19 cell lines, see Supplementary Table 2 , available online). § One-way analysis of variance was used to test for differences between the four subgroups present in the cell lines, with P < .05 from a post hoc Dunett t test being considered statistically significant. All statistical tests were two-sided. 64 Brief Communication | JNCI Vol. 101, Issue 1 | January 7, 2009 Figure 1 . Immunological assessment of antigens in breast cancer cell anti-CD44 conjugated with R-phycoerythrin coupled to the cyanine dye lines representing the four intrinsic breast cancer subtypes. Data from Cy7 (PE-Cy7, clone HIB19; eBioscience). We used 7AAD (Sigma-Aldrich, two normal-like breast cancer cell lines, one basal-like cell line, one St Louis, MO) to assess viability. Only viable (7AAD negative) cells luminal cell line, and one HER2 (ERBB2)-positive cell line are shown. were analyzed for antigen expression ( red histograms ). Unstained cells Cell surface antigens on tumor cells were assessed individually by incu- ( black histograms ) were used as a negative control. Data are from one bating 10 cultured human breast cancer cells with the following representative experiment that was performed three times. Results fl uorochrome-conjugated monoclonal antibodies: anti-EpCAM conju- were similar for all three experiments. EpCAM = epithelial cell adhe- gated with FITC (clone EBA-1; BD Biosciences, San Jose, CA), anti-CD24 sion molecule; FITC = fl uorescein isothiocyanate; 7AAD = 7-amino- conjugated with FITC (clone SN3; eBioscience, Inc., San Diego, CA), or actinomycin D. of hematopoietic origin are mucin 1 and expression of caveolin 1 (Spearman r =  0.26, clinical use, not only in cultured cell lines caveolin 1. Gene transcripts of caveolin 1, P < .001). Consequently, antibodies against but also in clinical samples of known in - an approximately 22-kDa integral mem- antigens such as caveolin 1 might be able to trinsic breast cancer subtype, as described brane protein, and mucin 1, a 350-kDa specifi cally detect circulating tumor cells previously for assays relying only on anti- glycoprotein that protects the cell surface, from normal-like breast tumors. EpCAM antibodies ( 5 ). were ubiquitously expressed in all normal- A limitation of this study is our use of In conclusion, an EpCAM-dependent like human breast tumor cell lines, with the homogeneous breast cancer cell lines of assay could not detect normal-like breast apparent expression of caveolin 1 being known subtypes instead of blood samples tumor cells. In the future, the identifi cation higher (gene transcript expression range = from patients with breast cancer that had of antibodies that specifi cally detect nor- 1.42 – 13.42, relative to the expression of been subtyped. Detection of normal-like mal-like breast cancer cells (which in gen- HMBS , HPRT1 , and GUSB ) than that of breast cancer cells in clinical (breast) cancer eral have aggressive features) and their mucin 1 (gene transcript expression range = samples, which consist of heterogeneous inclusion in the CellSearch assay may 0.06 – 0.56, relative to the expression of cell populations, will require an assay that improve the sensitivity and feasibility of HMBS , HPRT1 , and GUSB ) ( Table 1 ; uses a mixture of antibodies against differ- that assay without a loss of specifi city. Supplementary Table 3 , available online). ent cell surface antigens that are present on Furthermore, in our 344 clinical breast circulating tumor cells but absent on cells References cancer samples ( 17 ), decreased expression of hematopoietic origin. Such an assay 1. 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October 7 , 2008 ; accepted October 20 , 2008 . 66 Brief Communication | JNCI Vol. 101, Issue 1 | January 7, 2009 http://www.deepdyve.com/assets/images/DeepDyve-Logo-lg.png JNCI Journal of the National Cancer Institute Pubmed Central

Anti-Epithelial Cell Adhesion Molecule Antibodies and the Detection of Circulating Normal-Like Breast Tumor Cells

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Pubmed Central
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© 2008 The Author(s).
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0027-8874
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1460-2105
DOI
10.1093/jnci/djn419
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Abstract

BRIEF COMMUNICATION preparation system), the CellSearch epithe- Anti-Epithelial Cell Adhesion Molecule lial cell kit (to enrich for cells expressing Antibodies and the Detection of EpCAM and to label nuclei, leukocytes, and epithelial cells), and the CellSpotter Circulating Normal-Like Breast Tumor Analyzer (a semiautomated fl uorescence- based microscopy system that permits Cells computer-generated reconstruction of cel- lular images). By use of the CellSearch Anieta M. Sieuwerts , Jaco Kraan , Joan Bolt , Petra van der Spoel , Fons Elstrodt , epithelial cell kit, circulating tumor cells Mieke Schutte , John W. M. Martens , Jan-Willem Gratama , Stefan Sleijfer , John are isolated with anti-EpCAM antibodies A. Foekens coupled to microscopic iron particles, and Identification of specific subtypes of circulating tumor cells in peripheral blood of complexes of circulating tumor cells bound cancer patients can provide information about the biology of metastasis and to anti-EpCAM – coupled iron particles are improve patient management. However, to be effective, the method used to iden- “pulled” out of the blood sample by use of tify circulating tumor cells must detect all tumor cell types. We investigated whether powerful magnets. Unbound cells and the the five subtypes of human breast cancer cells that have been defined by global remaining plasma are aspirated, and the gene expression profiling — normal-like, basal, HER2-positive, and luminal A and immunomagnetically isolated cells are per- B — were identified by CellSearch, a US Food and Drug Administration – approved meabilized and stained with 4 ′ ,6-diamidino- test that uses antibodies against the cell surface – expressed epithelial cell adhesion 2-phenylindole (to detect nuclei); molecule (EpCAM) to isolate circulating tumor cells. We used global gene expres- anti-CD45 antibodies labeled with allophy- sion profiling to determine the subtypes of a well-defined panel of 34 human breast cocyan (to detect leukocytes); and anti- cancer cell lines (15 luminal, nine normal-like, five basal-like, and five Her2-positive). cytokeratin 8, 18, and 19 antibodies labeled We mixed 50-150 cells from 10 of these cell lines with 7.5 mL of blood from a single with phycoerythrin (to detect epithelial healthy human donor, and the mixtures were subjected to the CellSearch test to cells). After incubation with staining isolate the breast cancer cells. We found that the CellSearch isolation method, reagents, the magnetic separation is which uses EpCAM on the surface of circulating tumor cells for cell isolation, did repeated and excess staining reagents are not recognize, in particular, normal-like breast cancer cells, which in general have removed by aspiration. In the fi nal process- aggressive features. New tests that include antibodies that specifically recognize ing step, the immunomagnetically isolated normal-like breast tumor cells but not cells of hematopoietic origin are needed. cells are resuspended in a MagNest Cell Presentation Device (Veridex LLC). This J Natl Cancer Inst 2009;101: 61 – 66 device consists of a chamber and two mag- nets that orient the immunomagnetically Circulating tumor cells are cells that have However, it is unclear whether such tests labeled cells for analysis by use of the detached from the primary tumor or meta- can detect all tumor subtypes. We investi- static tumor sites and entered the periph- gated whether the CellSearch test could eral circulation. A limited number of recognize all subtypes of breast cancer, Affiliations of authors: Department of Medical markers are used for the isolation (ie, cell including normal-like, basal, HER2- Oncology, Josephine Nefkens Institute and Cancer surface antigens) or detection (ie, various positive, and luminal A and B tumor cells. Genomics Centre (AMS, JB, JWMM, JAF), Department of Medical Oncology, Daniel den antigens or mRNAs) of circulating tumor The CellSearch circulating tumor cell Hoed Cancer Center (JK, PvdS, J-WG, SS), and cells. These markers include epithelial cell test is the only diagnostic test that is cur- Department of Medical Oncology, Josephine surface markers, such as the epithelial cell rently approved by the US Food and Drug Nefkens Institute (FE, MS), Erasmus MC, Rotterdam, the Netherlands . adhesion molecule (EpCAM; also known as Administration as an automated test to Correspondence to: Anieta M. Sieuwerts, PhD, CD326, ESA, HEA125, and TACSTD1); detect and enumerate circulating tumor Department of Medical Oncology, Josephine cytokeratins 7, 8, 18, 19, and 20; and more cells ( 4 ). Briefl y, a blood sample that con- Nefkens Institute, Erasmus MC, Rm BE-400, Dr cancer-specific markers, such as HER2- tains many leukocytes and few circulating Molewaterplein 50, 3015 GE Rotterdam, the Netherlands (e-mail: a.sieuwerts@erasmusmc.nl ). neu and mucin 1 for breast carcinoma tumor cells is drawn into 10-mL CellSave See “Funding” and “Notes” following “References.” ( 1 – 3 ). Commercially available tests for iso- Preservative Tubes (Veridex LCC), which DOI: 10.1093/jnci/djn419 lation and detection of circulating tumor contain EDTA as an anticoagulant and a © 2008 The Author(s). cells include the CellSearch circulating cellular preservative. The blood is main- This is an Open Access article distributed under tumor cell test (Veridex LLC, San Diego, tained at room temperature and subse- the terms of the Creative Commons Attribution CA) and other tests ( 1 – 3 ). These tests use quently processed within 72 hours of Non-Commercial License (http://creativecommons. org/licenses/by-nc/2.0/uk/), which permits unre- combinations of specific antibodies against collection by use of the CellSearch system stricted non-commercial use, distribution, and these molecules and generally include anti- (Veridex LLC), which consists of the reproduction in any medium, provided the origi- bodies against EpCAM for cell isolation. CellTracks Autoprep (an automated sample nal work is properly cited. jnci.oxfordjournals.org JNCI | Brief Communication 61 patients who have responded to systemic chain reaction. The 14 candidate genes CONTEXT AND CAVEATS therapy (who have a decreased number of that may as such enable dis crimination Prior knowledge circulating tumor cells) from those who between the breast cancer subgroups Identification of specific subtypes of circu- have not (who have an increased number of included genes that are more specifi c for lating tumor cells in the peripheral blood of circulating tumor cells) ( 15 ). cells of hematopoietic origin ( CD44 and cancer patients can provide important prog- Five subtypes of human breast cancers CD45 ); epithelial cell – specifi c genes such nostic information, but to be effective, the have been identifi ed by global gene expres- as those encoding cytokeratins, EpCAM, method used must recognize all tumor cell sion profi ling, including normal-like, basal, and mucin 1; genes encoding markers types. HER2-positive, and luminal A and B ( 16 ). specifi c for the breast cancer subgroups Study design The normal-like and basal subtypes, which ( ESR1 , ERBB1 , ERBB2 , CAV1 , and CD24 ); The subtype of 19 well-characterized breast represented 7.8% and 25% of all breast and genes for two well-known epithelial – cancer cell lines was obtained by use of cancers, respectively, in a cohort of 344 mesenchymal transition markers ( TWIST1 gene expression profiling, including normal- breast tumor samples from patients with and VIM ). Transcript levels of these 14 like, basal-like, HER2-positive, and luminal lymph node – negative disease ( 17 ), are in genes in all the 34 cell lines were compared A and B. Cells from each line were mixed general negative for estrogen receptor with those measured in whole-blood cells with blood from a healthy donor and sub- ( ESR1 ), progesterone receptor, and HER2- from 23 different blood donors before (n = jected to the CellSearch circulating tumor neu, and so lack the molecular targets for 6 samples) and after (n = 23 samples) being cell assay. endocrine therapy and anti-HER2 – directed subjected to EpCAM-based CellSearch Contribution therapy and therefore have worse treat- enrichment to establish which genes were The CellSearch assay, which uses epithelial ment options than other subtypes. Because not expressed by blood cells and could be cell adhesion molecules on the cell surface, each breast cancer subtype has distinct used to specifi cally identify the epithelial did not recognize normal-like breast can - prognostic and therapeutic characteristics tumor cells ( Table 1 ). The study was cer cells, although other subtypes were ( 16 , 18 ), it is important to establish whether approved by the Erasmus MC Institutional recognized. each breast cancer subtype expresses the Review Board. Blood samples were col- Implications cell surface antigens that are used in assays lected from healthy volunteers after written Normal-like breast cancer cells have espe- to isolate and detect circulating tumor informed consent was obtained. Supple- cially aggressive features, and so assays cells. It is also important to identify which mentary Table 1 (available online) lists the that recognize this subtype would provide breast cancer subtypes do not express an gene expression assays used. valuable prognostic information. New antigen and to determine which antibodies We next randomly selected 19 of the 34 assays are needed that include antibodies could be used instead of or in addition to cell lines; incubated each cell line with that specifically recognize this breast can- cer subtype but not other cell types, includ- antibodies that are included in the test for fl uorochrome-conjugated antibodies against ing those of hematopoietic origin. circulating tumor cells. CD45, CD24, CD44, or EpCAM, which To investigate whether the CellSearch were chosen to enable a distinction between Limitations test recognized all fi ve breast cancer sub- CD45-positive cells of hematopoietic ori- Only homogeneous breast cancer cell lines types that are characterized by their intrin- gin, CD45-negative and EpCAM-positive with known subtypes were investigated. sic gene expression profi les as previously breast cancer cells, and CD45-negative, From the Editors described ( 16 , 18 ), we fi rst determined the CD24-negative, and CD44-positive breast subtypes of our well-defi ned panel of 34 cancer stem cells ( 21 ); and used fl ow cytom- CellSpotter Analyzer, a four-color semiau- human breast cancer cell lines ( 19 ) by etry to measure the expression of each tomated fl uorescence microscope. Finally, global gene expression profi ling, as marker by each cell line ( Table 2 ). Next, an circulating tumor cells that are defi ned as described previously ( 17 ). A previous analy- aliquot (50 – 150 cells per aliquot) of each of nucleated cells with a round to oval mor- sis of nearly 150 polymorphic microsatellite these 19 cell lines was added to a separate phology that are positive for anti- markers in this panel of 34 cell lines has 7.5-mL sample of peripheral blood from a cytokeratin antibody binding and negative shown that each of these 34 cell lines is single healthy volunteer donor, and the for anti-CD45 antibody binding ( 5 ) are unique and monoclonal ( 20 ). As determined mixtures were subjected to the EpCAM- identifi ed by an operator. by the expression of genes in the intrinsic based CellSearch assay to isolate circulat - Results of the CellSearch test have been gene set ( 16 , 17 ), the complete panel con- ing tumor cells. Gene expression data of used to monitor disease progression and tained 15 luminal cell lines (ie, no clear-cut the isolated cells showed that cells with a therapy effi cacy in metastatic prostate ( 6 ), discrimination between luminal A and B), luminal or HER2-positive subtype were colorectal ( 7 ), and breast ( 8 ) cancer. For nine normal-like lines, fi ve basal-like lines, generally isolated by the assay, whereas patients with advanced breast cancer, the and fi ve HER2-positive cell lines ( Table 1 ). cells with a normal-like subtype, which lack number of circulating tumor cells as deter- To validate the gene expression profi ling EpCAM expression, were not isolated or mined by the CellSearch test has been data, transcript levels of 14 candidate genes were only partially isolated (eg, MDA-MB- shown to have prognostic value ( 8 – 14 ). In were remeasured with Affymetrix GeneChip 231 cells, which have marginal EpCAM addition, a change from the baseline num- Exon 1.0 ST Arrays (Affymetrix UK Ltd., expression) ( Figure 1 ; Supplementary ber of circulating tumor cells after the fi rst Wickham la Wooburn Grn, UK) and Table 2 , available online). We found that cycle of therapy appears to distinguish real-time reverse transcriptase – polymerase the cell lines with the normal-like subtype 62 Brief Communication | JNCI Vol. 101, Issue 1 | January 7, 2009 jnci.oxfordjournals.org JNCI | Brief Communication 63 Table 1 . mRNA expression in breast cancer cell lines with different intrinsic subtype characteristics * Relative mRNA expression (95% CI) Relative mRNA expression (95% CI) Blood before Blood after Normal-like Basal-like Luminal HER2-positive enrichment enrichment Gene symbol (antigen) (n = 9) (n = 5) (n = 15) (n = 5) P † (n = 6) (n = 23) PTPRC (CD45) 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) .210 13.39 (10.42 to 16.37) 6.25 (4.99 to 7.50) CD24 (CD24) 0.40 (0.03 to 0.78) 2.31 (0.20 to 4.42) 3.84 (2.14 to 5.54) 2.69 (1.86 to 3.52) .025 0.03 (0.02 to 0.03) 0.03 (0.01 to 0.06) CD44 (CD44) 0.77 (0.59 to 0.95) 0.75 (0.40 to 1.10) 0.04 (0.02 to 0.06) 0.02 (0.00 to 0.04) <.001 3.85 (3.13 to 4.57) 3.90 (3.29 to 4.52) TACSTD1 (EpCAM) 0.04 (0.00 to 0.10) 2.04 (0.83 to 3.25) 8.72 (4.02 to 13.43) 4.43 (2.17 to 6.69) .020 0.00 0.01 (0.00 to 0.02) MUC1 (EMA, mucin 1) 0.32 (0.20 to 0.44) 0.35 (0.15 to 0.55) 1.88 (1.24 to 2.52) 0.53 (0.43 to 0.63) <.001 0.03 (0.02 to 0.05) 0.01 (0.00 to 0.02) CAV1 (caveolin 1) 5.04 (2.39 to 7.70) 1.70 (0.19 to 3.21) 0.05 (0.02 to 0.08) 0.03 (0.01 to 0.05) <.001 0.02 (0.01 to 0.04) 0.05 (0.00 to 0.01) KRT7 (cytokeratin 7) 1.53 (0.00 to 3.07) 7.90 (5.63 to 10.17) 6.62 (3.62 to 9.63) 10.46 (6.45 to 14.48) .009 0.01 (0.00 to 0.02) 0.01 (0.00 to 0.01) KRT18 (cytokeratin 18) 5.58 (0.96 to 10.19) 10.06 (7.88 to 12.24) 16.80 (13.23 to 20.36) 17.17 (12.11 to 22.23) .001 0.06 (0.03 to 0.09) 0.03 (0.02 to 0.03) KRT19 (cytokeratin 19) 0.09 (0.00 to 0.26) 4.53 (0.00 to 9.11) 28.35 (12.66 to 44.03) 11.99 (1.58 to 22.40) .022 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) ESR1 (estrogen receptor) 0.00 (0.00 to 0.00) 0.02 (0.01 to 0.03) 0.76 (0.37 to 1.14) 0.06 (0.00 to 0.16) .003 0.02 (0.01 to 0.03) 0.01 (0.00 to 0.02) ERBB1 (EGF receptor) 0.20 (0.10 to 0.30) 0.96 (0.00 to 2.01) 0.03 (0.01 to 0.05) 0.07 (0.00 to 0.14) .003 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) ERBB2 (HER2) 0.05 (0.03 to 0.06) 0.07 (0.02 to 0.12) 2.53 (0.13 to 4.94) 7.69 (2.28 to 13.09) .009 0.02 (0.01 to 0.03) 0.01 (0.00 to 0.01) TWIST1 (EMT marker TWIST1) 0.06 (0.01 to 0.11) 0.00 (0.00 to 0.00) 0.01 (0.00 to 0.02) 0.00 (0.00 to 0.00) .027 0.00 (0.00 to 0.00) 0.00 (0.00 to 0.00) VIM (EMT marker vimentin) 101.56 (36.90 to 116.23) 0.43 (0.00 to 0.90) 0.30 (0.00 to 0.67) 0.10 (0.00 to 0.27) <.001 34.47 (22.12 to 46.82) 52.30 (44.10 to 60.50) * Data in this table are the average and its 95% CI of one representative experiment. Results from other experiments were similar. Expression data are presented relative to that of HMBS , HPRT1 , and GUSB. Fourteen candidate genes were selected, including genes that are more specific for cells of hematopoietic origin ( CD44 and CD45 ); epithelial cell – specific genes such as those encoding cytokeratins, EpCAM, and mucin 1; genes encoding markers specific for the breast cancer subgroups ( ESR1 , ERBB1 , ERBB2 , CAV1 , and CD24 ); and genes for two well-known EMT markers ( TWIST1 and VIM ). Data of the individual cell lines are presented in Supplementary Table 3 (available online), and the gene expression assays used are shown in Supplementary Table 1 (available online). To validate the gene expression profiling data, transcript levels of candidate genes that might enable discrimination among the four breast cancer subtypes, as represented by 19 randomly selected breast cancer cell lines, were analyzed with Affymetrix GeneChip Exon 1.0 ST Arrays (Affymetrix UK Ltd., Wickham la Wooburn Grn, UK) and real-time RT-PCR and compared with levels measured in whole blood of healthy blood donors before (n = 6) or after (n = 23) EpCAM-based CellSearch enrichment for circulating tumor cells. RNA was isolated from healthy blood and cell lines with the RNeasy (Micro) kit (Qiagen BV, Venlo, the Netherlands). For the cell lines, cDNA was prepared by use of the Superscript II RNase H-kit from Invitrogen (Breda, the Netherlands). For healthy blood donors before CellSearch enrichment and the preparations from healthy blood donors and cell lines after CellSearch enrichment, cDNA was synthesized with the RevertAid H Minus First Strand cDNA synthesis kit (Fermentas, St Leon-Rot, Germany), followed by a linear preamplification for the non-[2-[ N -(3-dimethylaminopropyl)- N -propylamino]-4-[2,3-dihydro-3-methyl-(benzo-1,3-thiazol-2-yl)-methylidene]-1-phenyl-quinolinium] (SYBR Green I)-based assays (TaqMan PreAmp from Applied Biosystems, Nieuwerkerk a/d IJssel, the Netherlands), according to the manufacturer ’ s instructions. The resulting cDNA preparations were analyzed by real-time PCR with TaqMan gene expression assays and TaqMan Universal PCR Master Mix No AmpErase UNG (Applied Biosystems) and Absolute qPCR SYBR Green I mixture (Abgene, Epsom, UK) for the SYBR-based assays. PCRs were performed in a 20- µ L reaction volume in an Mx3000P Real-Time PCR system (Stratagene, Amsterdam, the Netherlands). Expression of HMBS , HPRT1 , and GUSB was used as a reference to control sample loading and RNA quality, as described previously (26). To enable comparison between the different methods of measuring gene expression (Affymetrix GeneChip Exon 1.0 ST arrays and real-time RT-PCR), data were normalized by expression levels that were measured by both methods in a set of seven control cell lines (ie, MDA-MB-435s, MDA-MB-231, CAMA-1, MCF-7, T47D, SK-BR-3, and EVSA-T). EGF = epidermal growth factor receptor; EMA = epithelial membrane antigen; EpCAM = epithelial cell adhesion molecule; EMT = epithelial – mesenchymal transition; CI = confidence interval; RT-PCR = reverse transcriptase – polymerase chain reaction; cDNA = complementary DNA. † One-way analysis of variance was used to test for differences between the four subgroups present in the cell lines, with P < .05 being considered statistically significant. All statistical tests were two-sided. expressed high mRNA levels of the epithe- to enter the circulation and seed metastases vimentin and CD44 mRNA and the low lial – mesenchymal transition markers, ( 23 – 25 ). Thus, the normal-like breast can- expression of CD24 mRNAs. Because anti- TWIST1 ( TWIST1 ) and vimentin ( VIM ) cer cell subtype is an important target for bodies against vimentin and CD44 also (one-way analysis of variance, two-sided the development of individualized therapy bind to cells of hematopoietic origin, these P = .03 and P < .001, respectively) ( Table 1 ); and should not be overlooked when assess- antibodies would not be suitable for the however, MDA-MB-231 cells expressed ing circulating tumor cells. Intrinsic breast detection of circulating tumor cells that vimentin but did not appear to express cancer subtypes are easily missed in analy- have an epithelial – mesenchymal transition TWIST1 ( Supplementary Table 3 , avail- ses that use only crude, standard clinical or origin or putative breast cancer stem cells. able online). pathological criteria such as hormone However, current strategies for detecting Cells with a normal-like breast cancer receptor and HER2 status. Therefore, our circulating tumor cells can be improved to subtype express high levels of genes that results, which are based on a distinction in allow assessment of these normal-like cells are characteristic of basal epithelial and breast cancer subtypes determined by by selecting antigens that are ubiquitously adipose cells and low levels of genes that global gene expression profi ling, cannot be and abundantly present and that can be are characteristic of luminal epithelial cells compared with those of Cristofanilli et al. made accessible for isolation with immuno- ( 16 ). The normal-like breast cancer subtype ( 8 ), who did not fi nd differences in the beads on normal-like cells but that are is the only subtype that displays the puta- number of circulating tumor cells between absent on cells of hematopoietic origin. tive tumor-initiating stem cell phenotype, breast cancer cell subtypes that were defi ned When selecting antigens to use for isolat- which includes low expression of CD24 only by hormone receptor and HER2 ing circulating normal-like breast cancer and high expression of CD44 ( 21 , 22 ) status. cells, it must be emphasized that clinical ( Table 2 ) and has high expression of New tests that include antibodies that (breast) cancer samples consist of heteroge- both vimentin and TWIST1 ( Table 1 ). specifi cally recognize normal-like breast neous cell populations and always contain Expression of vimentin and TWIST1 has tumor cells but not cells of hematopoietic leukocytes and other types of blood cells; been used to identify cells that have under- origin are needed. As shown in Table 1 , the we circumvented this problem by using gone the epithelial – mesenchymal transi- phenotype of circulating leukocytes in the homogeneous cell lines. Examples of mem- tion, a process that has been linked to the healthy blood donors before and after brane antigens that might fulfi ll the criteria generation of cells with properties of stem EpCAM-based CellSearch enrichment was of being ubiquitously and abundantly pres- cells and to the ability of breast cancer cells characterized by the high expression of ent on normal-like cells but absent on cells Table 2 . Immunological assessment of antigens in breast cancer cell lines with different intrinsic subtype characteristics and circulating tumor cell recovery * Flow cytometry, † MFI CellSearch, ‡ % of cells Intrinsic subtype No. of cell lines CD45 CD24 CD44 EpCAM recovered (95% CI) Normal-like 6 <5 <5 >1000 <5 2 (0 to 6) § Basal-like 5 <5 5– 20 200 – 1000 20 – 200 48 (36 to 61) Luminal 5 <5 5– 20 5 – 20 20 – 200 75 (62 to 89) HER2-positive 3 <5 5– 20 <5 20 – 200 85 (61 to 108) * MFI = mean fluorescence intensity; EpCAM = epithelial cell adhesion molecule; CI = confidence interval; 7AAD = 7-amino-actinomycin D; FITC = fluorescein isothiocyanate. † Cultured human breast cancer cell lines were incubated with the following fluorochrome-conjugated monoclonal antibodies: EpCAM conjugated with FITC (clone EBA-1; BD Biosciences, San Jose, CA), CD24 conjugated with FITC (clone SN3; eBioscience, Inc., San Diego, CA), CD44 conjugated with R-phycoerythrin coupled to the cyanine dye Cy7 (PE-Cy7, clone HIB19; eBioscience), and CD45 conjugated with allophycocyanin (clone 2D1; BD Biosciences). We used 7AAD (Sigma-Aldrich, St Louis, MO; 1 µ g/mL) to control for viability. Cells were then analyzed on a Canto flow cytometer (BD Biosciences). To exclude nonviable cells from analysis, only 7AAD-negative (viable) cells were evaluated for antigen expression. Unstained cells were used as a negative control. Only cells that expressed EpCAM were captured by the anti-EpCAM antibody present in the CellSearch circulating tumor cell profile kit. In addition, as shown in Figure 1 and Supplementary Table 2 (available online), MDA-MB-231 cells with marginal EpCAM expression were only partially (10 of 82 added tumor cells, or approximately 12%) captured by the CellSearch antibody, indicating that anti-EpCAM antibodies from the EBA-1 clone (BD Biosciences) and from the CellSearch circulating tumor cell kit have similar sensitivities and specificities. ‡ Data are the average (95% CI) of one representative experiment performed in duplicate for the given number of cell lines. Blood samples containing EDTA (7.5-mL aliquots of blood) from a single healthy blood donor were obtained from CellSave Preservative Tubes (Veridex LCC, San Diego, CA). To each sample, 20 µ L of a cell suspension containing 50 – 150 cultured cells from the indicated subtype of human breast cancer was added. To determine the actual viable cell number, a 100- µ L aliquot of the cultured cells was incubated with 10 µ L of 7AAD (1 µ g/mL) and 100 µ L of fluorescent beads (Beckman – Coulter, Inc., Miami, FL). After a 15-minute incubation at room temperature, 2 mL of phosphate-buffered saline was added, and samples were analyzed on a Calibur flow cytometer (BD Biosciences). At least 10 000 beads were acquired to estimate the number of 7AAD-negative (viable) cells. The efficiency of retrieving the tumor cells was controlled by counting the exact number of viable cells that were drawn in triplicate by flow cytometry and by light microscopy after serial dilution. To establish the number of circulating tumor cells recovered, samples were processed on the CellTrack AutoPrep analyzer (Veridex LLC) with the CellSearch circulating tumor cell profile kit (Veridex LLC), which uses microscopic iron particles coupled to anti-EpCAM to enrich for circulating tumor cells. The number of circulating tumor cells (ie, cells stained with the nuclear dye, 4 ′ ,6-diamidino-2-phenylindole, that are positive for cytokeratin 8, 18, and 19, and negative for CD45) were determined on the CellSpotter analyzer (Veridex LLC), according to the manufacturer’s instructions (for full flow cytometry and CellSearch recovery data on these 19 cell lines, see Supplementary Table 2 , available online). § One-way analysis of variance was used to test for differences between the four subgroups present in the cell lines, with P < .05 from a post hoc Dunett t test being considered statistically significant. All statistical tests were two-sided. 64 Brief Communication | JNCI Vol. 101, Issue 1 | January 7, 2009 Figure 1 . Immunological assessment of antigens in breast cancer cell anti-CD44 conjugated with R-phycoerythrin coupled to the cyanine dye lines representing the four intrinsic breast cancer subtypes. Data from Cy7 (PE-Cy7, clone HIB19; eBioscience). We used 7AAD (Sigma-Aldrich, two normal-like breast cancer cell lines, one basal-like cell line, one St Louis, MO) to assess viability. Only viable (7AAD negative) cells luminal cell line, and one HER2 (ERBB2)-positive cell line are shown. were analyzed for antigen expression ( red histograms ). Unstained cells Cell surface antigens on tumor cells were assessed individually by incu- ( black histograms ) were used as a negative control. Data are from one bating 10 cultured human breast cancer cells with the following representative experiment that was performed three times. Results fl uorochrome-conjugated monoclonal antibodies: anti-EpCAM conju- were similar for all three experiments. EpCAM = epithelial cell adhe- gated with FITC (clone EBA-1; BD Biosciences, San Jose, CA), anti-CD24 sion molecule; FITC = fl uorescein isothiocyanate; 7AAD = 7-amino- conjugated with FITC (clone SN3; eBioscience, Inc., San Diego, CA), or actinomycin D. of hematopoietic origin are mucin 1 and expression of caveolin 1 (Spearman r =  0.26, clinical use, not only in cultured cell lines caveolin 1. Gene transcripts of caveolin 1, P < .001). Consequently, antibodies against but also in clinical samples of known in - an approximately 22-kDa integral mem- antigens such as caveolin 1 might be able to trinsic breast cancer subtype, as described brane protein, and mucin 1, a 350-kDa specifi cally detect circulating tumor cells previously for assays relying only on anti- glycoprotein that protects the cell surface, from normal-like breast tumors. EpCAM antibodies ( 5 ). were ubiquitously expressed in all normal- A limitation of this study is our use of In conclusion, an EpCAM-dependent like human breast tumor cell lines, with the homogeneous breast cancer cell lines of assay could not detect normal-like breast apparent expression of caveolin 1 being known subtypes instead of blood samples tumor cells. In the future, the identifi cation higher (gene transcript expression range = from patients with breast cancer that had of antibodies that specifi cally detect nor- 1.42 – 13.42, relative to the expression of been subtyped. Detection of normal-like mal-like breast cancer cells (which in gen- HMBS , HPRT1 , and GUSB ) than that of breast cancer cells in clinical (breast) cancer eral have aggressive features) and their mucin 1 (gene transcript expression range = samples, which consist of heterogeneous inclusion in the CellSearch assay may 0.06 – 0.56, relative to the expression of cell populations, will require an assay that improve the sensitivity and feasibility of HMBS , HPRT1 , and GUSB ) ( Table 1 ; uses a mixture of antibodies against differ- that assay without a loss of specifi city. Supplementary Table 3 , available online). ent cell surface antigens that are present on Furthermore, in our 344 clinical breast circulating tumor cells but absent on cells References cancer samples ( 17 ), decreased expression of hematopoietic origin. Such an assay 1. 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October 7 , 2008 ; accepted October 20 , 2008 . 66 Brief Communication | JNCI Vol. 101, Issue 1 | January 7, 2009

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JNCI Journal of the National Cancer InstitutePubmed Central

Published: Jan 7, 2009

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