| | Mammaglobin-A is a tumor-associated antigen in human breast carcinoma☆☆☆★Accepted 7 June 2002. Abstract Background. Mammaglobin-A is an attractive target for immune-based therapy for patients with breast cancer because of its exclusive expression in breast cancer. In this study, we attempted to identify immunogenic T cell epitopes restricted by human leukocyte antigen (HLA)-A2 in mammaglobin-A protein. Methods. To identify HLA-A2-restricted immunogenic epitopes from mammaglobin-A, 7 candidate peptides were synthesized and tested for immunogenicity. Each peptide was tested for binding to HLA-A2 in a HLA-A2 stabilization assay. Furthermore, T lymphocytes from 7 healthy donors and 1 patient with breast cancer received 3 weekly stimulations with autologous peptide-pulsed dendritic cells. Stimulated T cells were tested for specific recognition of peptide and tumor cells by interferon-γ enzyme-linked immunosorbent assay. Results. HLA-A2 binding assays showed that all designed peptides could bind to HLA-A2. Two of the 7 peptides (MAM3 and MAM7) successfully induced peptide-specific T cells. However, only MAM3-specific T cells recognized the mammaglobin overexpressing breast cancer cell line, MDA415 transfected with HLA-A2. In contrast, MAM3-specific T cell did not recognize wild type MDA415 or MDA415 transfected with HLA-A24, or the mammaglobin negative, HLA-A2 positive breast cancer cell line, MCF-7. Conclusions. Mammaglobin-A-derived peptide, MAM3, can induce mammaglobin-A-specific immunity and could be useful for vaccine strategies for patients with breast cancer. (Surgery 2003;133:74-80.)
Recent advances in tumor immunology have elucidated several crucial mechanisms of tumor cell recognition by human T cells. It has become clear that tumor-specific CD8+ T cells recognize tumor antigen-derived peptides, which usually consist of 8 to 10 amino acids, and the peptide presenting major histocompatibility complex (MHC) class I molecules.1 These peptides are generated by digesting antigenic proteins in cytosolic areas of tumor cells and loaded onto MHC class I molecules in the endoplasmic reticulum.2, 3 The first T cell epitopes were identified in melanoma through screening of a melanoma-derived complementary DNA expression library by melanoma-specific T cell clones.4, 5, 6 Since then, numerous epitopes have been identified in a variety of cancers.7 Clinical testing of peptide-based vaccines has demonstrated successful immunization and significant clinical responses in selected patients.8
Another important development has been a drastically improved understanding of dendritic cell (DC) biology.9 As professional antigen presentation cells, DCs have been shown to induce strong T cell immune responses against tumor. DCs in combination with antigenic epitopes have the ability to induce epitope-specific T cells from peripheral blood lymphocytes and overcome tolerance or ignorance, 2 phenomena that have been associated with poor T cell responses to tumor.10 Current knowledge on amino acid motifs required to bind certain MHC molecules provides a powerful tool to predict potential antigenic epitopes from target proteins.11 Therefore, epitope candidates can be artificially synthesized and evaluated for their ability to induce specific T cells that recognize tumor cells by stimulation of T cells with peptide-pulsed autologous DCs. Most antigenic epitopes of over-expressed genes in relatively weak immunogenic tumors, such as carcinoembryonic antigen,12 prostate-specific antigen,13 mucin-1,14 and Her2/ neu15 expressed in a variety of epithelial tumors, have been identified through this strategy, so-called “reverse immunology.”
In this study, we focused on mammaglobin-A as a possible tumor antigen in breast cancer. The mammaglobin-A gene encodes a 10-kDa glycoprotein that is distantly related to a family of epithelial secretory proteins.16 The mammaglobin-A gene is exclusively expressed in breast tissue. Moreover, about 80% of breast cancers overexpress mammaglobin-A protein.17 Hence, mammaglobin-A is expected to be a potential tumor antigen. We successfully identified a significant immunogenic epitope from mammaglobin-A by using reverse immunology and demonstrated that mammaglobin-A might be an attractive target for future immune therapy for breast cancer.
Material and methods  Peptide synthesis Peptides were purchased from Research Genetics (Huntsville, Ala). Synthesis was carried out by standard solid-phase method on the basis of fluorenylmethoxycarbonyl chemistry. Recovered lyophilized peptide was purified by high-pressure liquid chromatography on C-18 columns and peptide identity and purity (>95%) was demonstrated by mass spectrometry. Cultured cell lines Human leukocyte antigen (HLA)-A2+ peptide transporter associated protein-deficient T-B cell hybrid T2 cell line, and the breast cancer cell lines, MCF-7, HS578T, MDA361, MDA415, MDA435, MDA436, MDA465, MDA468, T47D, and ZR75-1 were purchased from the American Type Culture Collection (Manassas, Va). All lines were cultured at Roswell Park Memorial Institute (RPMI) 1640 (Mediatech, Herndon, Va), supplemented with 10% heat-inactivated fetal bovine serum (Sigma, St Louis, Mo), 1% glutamine (Mediatech), and 1% penicillin-streptomycin (Mediatech). Immunoblotting Gelelectrophoresis was carried out on 16% polyacrylamide gels by the method of Laemmli. Briefly, cells were lysed in buffer consisting of 1% Triton X-100, 10 mmol/L Tris (pH 7.4), 0.15 mol/L NaCl, 10 μg/mL aprotinin, 10 μg mL leupeptin, and 25 μg/mL phenylmethylsulfonyl fluoride for 5 minutes on ice. An aliquot was mixed with an equal amount of 2 × sample loading buffer and was denatured at 100°C for 5 minutes. Each lane was loaded with a lysate from 5 × 104 cells. After electrophoresis, proteins were transferred onto nitrocellulose membranes and probed with antimammaglobin-A polyclonal antibody and appropriate secondary antibodies. The blot was developed by chemiluminescence (Amersham, Arlington Heights, Ill). HLA-A2 transfection MDA415 cells were transduced by a phoenix retrovirus transduction system with the HLA-A2 retrovirus.18 In brief, 1 × 106 cells/well phoenix A packaging cells were seeded into a 6-well plate 24 hours before transfection. A transient transfection was performed using a Superfect transfection kit (Qiagen, Valencia, Calif) with the HLA-A2 retrovirus expression vector. Forty-eight hours after transfection, MDA415 cells were incubated with supernatant from the HLA-A2 transfected phoenix A packaging cells in the presence of 10 μg Polybrene (Sigma) and centrifuged at 2000 rpm for 20 minutes. The supernatant was replaced by fresh medium after 48 hours. The next day, a sample of the transfected cells was stained by anti-HLA-A2 monoclonal antibody (mAb), BB7.2, to evaluate cell surface expression of HLA-2. The positive cells were sorted by cell sorter, (Facs Vantage, Becton-Deckinson, San Jose, Calif) and cultured in growth medium with G418 (Gibco, Rockville, Md). Finally, the surface expression of HLA-class I, HLA-A2, and CD54 (intracellular adhesion molecule [ICAM]-1) was assessed by flow cytometry after indirect or direct immunofluorescence staining before and after interferon (IFN)-γ treatment (Endogen, Woburn, Mass). Antibodies used for indirect staining were the anti-HLA-A2 mAb, BB7.2, and anti-HLA class I, W6/32. Direct staining was performed with a specific mAb conjugated to fluorescein isothiocyanate: anti-ICAM fluorescein isothiocyanate (Pharmingen, San Jose, Calif). HLA-A2 stabilization assay T2 cells were kept in medium or pulsed with saturating amounts of individual peptides (100 μg/mL) for 12 hours and then stained with the HLA-A2-specific mAb, BB7.2, by indirect staining. Flow cytometry analysis, FACScaliber (Becton-Deckinson) was performed and the mean channel fluorescence was determined for each sample. Subsequently, the relative mean channel fluorescence was calculated as mean channel fluorescence after peptide pulsing divided by mean channel fluorescence without peptide pulsing. In vitro generation of DCs DCs were generated from peripheral blood mononuclear cells from healthy donors who had positive HLA-A2 findings or a patient with breast cancer by centrifugation on Histopaque (Sigma). Peripheral blood mononuclear cells were plated in 10-cm2 culture dishes (1 × 108 cells/ dish), and monocyte-enriched adherent cells were observed after a 4-hour incubation at 37°C. The nonadherent cells were removed and cryopreserved, and the adherent cells were cultured in the presence of 800 U/mL recombinant (r)-granulocyte-macrophage colony-stimulating factor (Endogen) and 400 U/mL recombinant interleukin-4 (Endogen) in AIM-V medium (Gibco). On day 6, 100 ng/mL lipopolysaccharide (Sigma) was added to the medium and on day 7, the cytokine-treated cells were harvested and used as mature DCs. In vitro cytotoxic lymphocyte induction using mammaglobin-A-derived peptides and DCs DCs were pulsed with saturating amounts of individual peptides (100 μg/mL) for 1 hour at 37°C. The peptide-loaded DCs were irradiated with 50 Gy and mixed with peripheral blood lymphocytes at a ratio of 1:10 in the presence of 50 U/mL rIL-2 in AIM-V medium with 2.5% human AB serum (Omega, Tarzana, Calif). On day 7, the responder cells were restimulated with peptide-loaded DCs in medium with 50 U/mL rIL-2. Responder cells received at least 3 weekly stimulations. Evaluation of antigen recognition by a T cell line and peptide-stimulated T cells Cytokine (IFN-γ) release assays were performed to evaluate the recognition of peptide and mammaglobin-A over-expressing tumor cells; 105 peptide-stimulated T cells were co-incubated with 105 tumor cells or peptide-loaded T2 cells for 24 hours at 37°C. The concentration of human IFN-γ in coculture supernatants was then determined using a commercially available enzyme-linked immunosorbent assay kit (Biosource, Camarillo, Calif). Results Selection and modification of target cell lines To investigate whether MAM3- and MAM7-specific T cells recognize HLA-A2+, mammaglobin-A-expressing breast cancer cells, mammaglobin-A protein expression was evaluated in 9 breast cancer cell lines by Western blot (Fig 2).
In spite of the high frequency of mammaglobin-positive breast cancers in vivo, mammaglobin-A protein was only detected in MDA415. Two additional breast cancer cell lines, the mammaglobin messenger RNA (mRNA) overexpressing cell line, EF192, 21 and the SV40 transformed breast cell line, BT-20, did not express mammaglobin-A protein (data not shown). Therefore, we focused on MDA415 to screen for recognition by MAM peptide-specific T cells. Because MDA415 expresses high levels of HLA class I (Fig 3, A) but not HLA-A2 (Fig 3, B) on its cell surface according to phenotype analysis, the HLA-A2 gene was transfected into MDA415 using a retrovirus encoding HLA-A2 (Fig 3, B).
MDA415-A2 was further tested for its expression level of surface adhesion molecule ICAM-1, which plays an important role in recognition of tumor cells by activated T cells.22 Neither MDA415 nor MDA415-A2 expressed ICAM-1; IFN-γ treatment, however, restored expression of ICAM-1 and upregulated HLA class I (Fig 3, A and C). Tumor recognition by peptide-specific T cells Finally, we tested tumor recognition by MAM3 and MAM7 peptide-specific T cell lines from 2 donors. All lines strongly recognized the peptide used for stimulation pulsed onto T2 cells (Table III). However, only MAM3-specific T cells recognized IFN-γ-treated MDA415-A2 (Fig 4).
Furthermore, MAM3-specific T cells did not recognize IFN-γ treated wild type MDA415 cells or MDA415 transfected with the gene for HLA-A24, MDA415-A24. The control, mammaglobin-A negative, HLA-A2+ breast cancer cell line, MCF-7 was not recognized, either ( Fig 4). On the other hand, MAM7-specific T cells failed to recognize any of the target cells tested. Discussion Several studies have demonstrated that mammaglobin-A is exclusively expressed in about 80% of breast cancers17 and can be used as a marker gene for the detection of micrometastasis.21, 23, 24, 25 Because of polymerase chain reaction-based methods, most of the studies focused on mRNA to detect mammaglobin expression.21 Immunohistochemical studies also suggest that about 80% of breast cancers overexpress mammaglobin-A gene products.17 Because of its expression profile, we hypothesized that mammaglobin-A could be a breast tumor-specific antigen and encode T cell epitopes. Unexpectedly, most breast cancer cell lines do not express mammaglobin-A protein. Although in vivo there is a clear correlation between mRNA and protein expression of mammaglobin-A,17 in vitro, most cell lines are protein-negative even when mRNA is readily detectable such as in MDA361, MDA468,16 and EF192.21 Recent studies suggest that natural mammaglobin-A forms a heterodimer with lipophilin B, and the dimerization is crucial for stabilization of mammaglobin-A protein.26 It is thought that either mammaglobin-A or lipophilin B gene expression is lost in vitro. Possibly unknown essential factors such as certain hormones may sustain gene expression of either mammaglobin-A or lipophilin B in vivo. However, in spite of extensive analysis of the promotor region of mammaglobin-A, its gene regulation is still unknown and remains an active part of our investigation.27 Using an established algorithm to predict binding to HLA-A2, 7 potentially high-binding peptides from the mammaglobin-A protein sequence were selected for study (Table I). The T2 stabilization assay showed that 6 of the 7 peptides bind HLA-A2 with high affinity and with relatively small differences between individual peptides (Fig 1). Thus, the algorithm generated from identified peptide binding motifs appears to have limitations to predict the exact binding affinity, perhaps because the binding affinity might be determined by both binding motif and the 3 dimensional structure of the peptide in the MHC groove.28 Similar discrepancies between predicted and actual binding affinity were found for other peptides, as well.29, 30 To screen T cells for recognition of mamaglobin-A-derived peptides presented by HLA-A2, the mammaglobin-A expressing breast cancer cell line, MDA415 was transfected with a retroviral vector expressing the HLA-A2 gene. In addition, because the costimulatory molecule, ICAM-1 (CD54) was absent on both wild type MDA415 and MDA415-A2, IFN-γ was used to restore expression. Although in general, in vitro manipulation may introduce artifacts, in our case we do not believe transfection introduced false-positive data for the following reasons: (1) the retroviral vector was tested by other investigators in a similar experimental setting and not found to introduce artifacts18; (2) the T cells were cultured for several weeks in the presence of mammaglobin peptide without exposure to transfected MDA415, thereby selecting for mammaglobin-specific T cells; and (3) all tested controls were negative. These included wild type MDA415, the mammaglobin-negative, HLA-A2-positive MCF7, and MDA415-A24. Even after treatment with IFN-γ these controls were not recognized (Fig 4). Only T cells sensitized with MAM3 recognized MDA415-A2. T cells from the same donor stimulated with the same DCs, but with a different peptide (MAM7) did not recognize MDA415-A2, even after treatment with IFN-γ. Both MAM3 and MAM7 induced strong T cell reactivity against T2 pulsed with each peptide. Only MAM3-specific T cells, however, recognized a mammaglobin-A+, HLA-A2+ breast cancer cell line. This result suggests that the MAM7 peptide cannot be expressed by breast cancer cells perhaps because it is not generated by the proteasome.3 It should be noted that MAM7 is encoded by the signal sequence region of the mammaglobin-A gene.16 The mammaglobin-A homologue, mammaglobin-B, which is expressed in several normal tissues other than breast has the same MAM7 sequence.31 Therefore, we conclude that of our selected peptides, MAM3 is the best candidate peptide for mammaglobin-based breast cancer-specific immune therapy. We recently obtained a MAM3-specific tetramer from the National Institutes of Health Tetramer Facility that would permit us to stain lymphocytes from patients with breast cancer and healthy donors for the presence of MAM3-specific T cells and determine their frequency through flow cytometric analysis. In conclusion, mammaglobin-A is a tumor antigen in human breast cancer. The antigenic epitope, MAM3, could play an important role in the development of future immune therapy for breast cancer. Acknowledgements The phoenix retrovirus transduction system was kindly provided by Dr Nolan, Stanford University, Palo Alto, Calif; the HLA-A2 retrovirus by Dr M. Sadelain, Memorial Sloan- Kettering Cancer Center, New York, NY; and rIL-2 by Amgen, Thousand Oaks, Calif. References 1.
1
Boon T, Cerottini JC, Van den Eynde B, van der Bruggen P, Van Pel A.
Tumor antigens recognized by T lymphocytes.
Annu Rev Immunol. 1994;12:337–365. MEDLINE 2.
2
York IA, Rock KL.
Antigen processing and presentation by the class I major histocompatibility complex.
Annu Rev Immunol. 1996;14:369–396. MEDLINE |
CrossRef
3.
3
Morel S, Levy F, Burlet-Schiltz O, Brasseur F, Probst-Kepper M, Peitrequin AL, et al.
Processing of some antigens by the standard proteasome but not by the immunoproteasome results in poor presentation by dendritic cells.
Immunity. 2000;12:107–117. MEDLINE |
CrossRef
4.
4
van der Bruggen P, Traversari C, Chomez P, Lurquin C, De Plaen E, Van den Eynde B, et al.
A gene encoding an antigen recognized by cytolytic T lymphocytes on a human melanoma.
Science. 1991;254:1643–1647. MEDLINE 5.
5
Kawakami Y, Eliyahu S, Sakaguchi K, Robbins PF, Rivoltini L, Yannelli JR, et al.
Identification of the immunodominant peptides of the MART-1 human melanoma antigen recognized by the majority of HLA-A2-restricted tumor infiltrating lymphocytes.
J Exp Med. 1994;180:347–352. MEDLINE |
CrossRef
6.
6
Bakker AB, Schreurs MW, de Boer AJ, Kawakami Y, Rosenberg SA, Adema GJ, et al.
Melanocyte lineage-specific antigen gp100 is recognized by melanoma-derived tumor-infiltrating lymphocytes.
J Exp Med. 1994;179:1005–1009. MEDLINE |
CrossRef
7.
7
Van den Eynde BJ, van der Bruggen P.
T cell defined tumor antigens.
Curr Opin Immunol. 1997;9:684–693. MEDLINE |
CrossRef
8.
8
Rosenberg SA, Yang JC, Schwartzentruber DJ, Hwu P, Marincola FM, Topalian SL, et al.
Immunologic and therapeutic evaluation of a synthetic peptide vaccine for the treatment of patients with metastatic melanoma.
Nat Med. 1998;4:321–327. MEDLINE |
CrossRef
9.
9
Banchereau J, Schuler-Thurner B, Palucka AK, Schuler G.
Dendritic cells as vectors for therapy.
Cell. 2001;106:271–274. MEDLINE |
CrossRef
10.
10
Smyth MJ, Godfrey DI, Trapani JA.
A fresh look at tumor immunosurveillance and immunotherapy.
Nat Immun. 2001;2:293–299. 11.
11
Kawashima I, Hudson SJ, Tsai V, Southwood S, Takesako K, Appella E, et al.
The multi-epitope approach for immuno-therapy for cancer: identification of several CTL epitopes from various tumor-associated antigens expressed on solid epithelial tumors.
Hum Immunol. 1998;59:1–14. MEDLINE |
CrossRef
12.
12
Alters SE, Gadea JR, Philip R.
Immunotherapy of cancer: generation of CEA specific CTL using CEA peptide pulsed dendritic cells.
Adv Exp Med Biol. 1997;417:519–524. MEDLINE 13.
13
Correale JP, Walmsley K, Zaremba S, Zhu M, Schlom J, Tsang KY.
Generation of human cytolytic T lymphocyte lines directed against prostate-specific antigen (PSA) employing a PSA oligoepitope peptide.
J Immunol. 1998;161:3186–3194. MEDLINE 14.
14
Domenech N, Henderson RA, Finn OJ.
Identification of an HLA-A11-restricted epitope from the tandem repeat domain of the epithelial tumor antigen mucin.
J Immunol. 1995;155:4766–4774. MEDLINE 15.
15
Peoples GE, Goedegebuure PS, Smith R, Linehan DC, Yoshino I, Eberlein TJ.
Breast and ovarian cancer-specific cytotoxic T lymphocytes recognize the same HER2/neu-derived peptide.
Proc Natl Acad Sci U S A. 1995;92:432–436. MEDLINE |
CrossRef
16.
16
Watson MA, Fleming TP.
Mammaglobin, a mammary-specific member of the uteroglobin gene family, is overexpressed in human breast cancer.
Cancer Res. 1996;56:860–865. MEDLINE 17.
17
Watson MA, Dintzis S, Darrow CM, Voss LE, DiPersio J, Jensen R, et al.
Mammaglobin expression in primary, metastatic, and occult breast cancer.
Cancer Res. 1999;59:3028–3031. MEDLINE 18.
18
Latouche JB, Sadelain M.
Induction of human cytotoxic T lymphocytes by artificial antigen-presenting cells.
Nat Biotechnol. 2000;18:405–409. MEDLINE |
CrossRef
19.
19
Gotch F, Rothbard J, Howland K, Townsend A, McMichael A.
Cytotoxic T lymphocytes recognize a fragment of influenza virus matrix protein in association with HLA-A2.
Nature. 1987;326:881–882. MEDLINE |
CrossRef
20.
20
Ikuta Y, Okugawa T, Furugen R, Nagata Y, Takahashi Y, Wang L, et al.
A HER2/NEU-derived peptide, a K(d)-restricted murine tumor rejection antigen, induces HER2-specific HLA-A2402-restricted CD8(+) cytotoxic T lymphocytes.
Int J Cancer. 2000;87:553–558. MEDLINE |
CrossRef
21.
21
Suchy B, Austrup F, Driesel G, Eder C, Kusiak I, Uciechowski P, et al.
Detection of mammaglobin expressing cells in blood of breast cancer patients.
Cancer Lett. 2000;158:171–178. Abstract | Full Text |
Full-Text PDF (101 KB)
|
CrossRef
22.
22
Tahara K, Sadanaga N, Nagashima H, Kitano S, Mori M.
MAGE-specific cytotoxic T lymphocytes require non-specific CD54 adherence receptors on carcinoma cells.
Int J Oncol. 2000;17:805–810. MEDLINE 23.
23
Min CJ, Tafra L, Verbanac KM.
Identification of superior markers for polymerase chain reaction detection of breast cancer metastases in sentinel lymph nodes.
Cancer Res. 1998;58:4581–4584. MEDLINE 24.
24
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 transcriptase polymerase chain reaction assay for mammaglobin mRNA.
J Clin Oncol. 1999;17:2015–2019. 25.
25
Manzotti M, Dell'Orto P, Maisonneuve P, Zurrida S, Mazzarol G, Viale G.
Reverse transcription-polymerase chain reaction assay for multiple mRNA markers in the detection of breast cancer metastases in sentinel lymph nodes.
Int J Cancer. 2001;95:307–312. MEDLINE |
CrossRef
26.
26
Colpitts TL, Billing-Medel P, Friedman P, Granados EN, Hayden M, Hodges S, et al.
Mammaglobin is found in breast tissue as a complex with BU101.
Biochemistry. 2001;40:11048–11059. 27.
27
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;16:817–824. MEDLINE 28.
28
Kuhns JJ, Batalia MA, Yan S, Collins EJ.
Poor binding of a HER-2/neu epitope (GP2) to HLA-A2.1 is due to a lack of interactions with the center of the peptide.
J Biol Chem. 1999;274:36422–36427. MEDLINE |
CrossRef
29.
29
Kessler JH, Beekman NJ, Bres-Vloemans SA, Verdijk P, van Veelen PA, Kloosterman-Joosten AM, et al.
Efficient identification of novel HLA-A(*)0201-presented cytotoxic T lymphocyte epitopes in the widely expressed tumor antigen PRAME by proteasome-mediated digestion analysis.
J Exp Med. 2001;193:73–88. MEDLINE |
CrossRef
30.
30
Tanaka Y, Amos KD, Joo HG, Eberlein TJ, Goedegebuure PS.
Modification of the HER2/NEU-derived tumor antigen GP2 improves induction of GP2-reactive cytotoxic T lymphocytes.
Int J Cancer. 2001;94:540–544. MEDLINE |
CrossRef
31.
31
Becker RM, Darrow C, Zimonjic DB, Popescu NC, Watson MA, Fleming TP.
Identification of mammaglobin B, a novel member of the uteroglobin gene family.
Genomics. 1998;54:70–78. MEDLINE |
CrossRef
Washington University School of Medicine, Department of Surgery, and Alvin J. Siteman Cancer Center, St Louis, Mo ☆ Supported by National Institutes of Health grant R01 CA68500. Dr Amos is an Ethicon-Society of University Surgeons Research Fellow. ☆☆ Reprint requests: Peter S. Goedegebuure, PhD, Washington University School of Medicine, Department of Surgery, Box 8109, 660 S Euclid Ave, St Louis, MO 63110. ★ 0039-6060/2003/$30.00 + 0 PII: S0039-6060(02)21692-X doi:10.1067/msy.2003.92 © 2003 Published by Elsevier Inc. | |
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