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Quantitative Fluorescence Cytometric Measurement of Estrogen and Progesterone Receptors: Correlation with the Hormone Binding Assay

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Abstract

We describe, here, a rapid flow cytometry technique for the detection and quantification of estrogen (ER) and progesterone (PgR) receptors in several human cell lines and in clinical samples obtained from breast cancer tumors. ER and PgR quantitation can be very useful in patients with breast cancer as their role in diagnosis and prognosis is well established. However ligand binding assays and immunohistochemical assays are difficult to measure heterogeneity in individual cells. On the other hand, flow cytometry is a convenient tool for quantification in individual cells. Flow cytometric results with breast cancer cell lines and clinical samples were compared to those obtained by quantitative biochemical ER and PgR performed by the standard dextran-coated charcoal biochemical assay. The latter assay is affected by the level of endogenous steroids. This is also the case in the routine measurement of ER/PgR in patient's tumor cells whereby estradiol molecules in patient's serum produced negative or low values in the biochemical assay. The mAbs used in our flow cytometric method bind to their specific ER or PgR independently of whether they are preoccupied by their ligands and they produce reliable results. With the use of beads calibrated in MESF (Molecules of Equivalent Soluble Fluorochrome) units, the ER and PgR can be measured on a per cell basis. The flow cytometric method showed a strong correlation with biochemical receptor assessments of either ERα (ERαDCC, r = 0.918, p = 0.073) or PgR (PgRDCC, r = 0.75, p = 0.001). This study demonstrates that ERα and PgR can be detected by flow cytometry on a per cell basis in intact cells, and can be quantitated reliably in terms of MESF without the limitations of competition with serum's estradiol molecules.

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References

  1. Early Breast Cancer Trialist's Collaborative Group: Tamoxifen for early breast cancer: an overview of the randomised trials. The Lancet 351: 1451-1467, 1998

    Google Scholar 

  2. Poola I, Williams DM, Koduri S, Ramprakash J, Taylor RE, Hankins WD: Quantitation of estrogen receptor mRNA copy numbers in breast cell lines and tumors flow cytometry: interlaboratory variation. Anal Biochem 258: 209-215, 1998

    Google Scholar 

  3. Sabe I, Andritsch I, Magoud A, Awad AS, Khalifa A, Krishan A: Flow cytometric analysis of estrogen receptor expression in isolated nuclei and cells from mammary cancer tissues. Cytometry 36: 131-139, 1999

    Google Scholar 

  4. Molino A, Micciolo R, Turazza M, Bonetti F, Piubello Q, Corgnati L, Martignoni G, Bonetti A, Nortilli R, Rodella S, Capelli P, Manfrin E, Pelosi G, Cetto GL: Estrogen receptors in699 primary breast cancers: a comparison of immunohistochemical and biochemical methods. Breast Cancer Res Treat 34: 221-228, 1995

    Google Scholar 

  5. Redkar A, Krishan A: Flow cytometric analysis of estrogen, progesterone receptor expression and DNA content in formalin fixed, paraffin-embedded human breast tumors. Cytometry 38: 61-69, 1999

    Google Scholar 

  6. Mote PA, Johnston JF, Manninen T, Tuohimaa P, Clarke CL: Detection of progesterone receptors forms A and B by immunohistochemical analysis. J Clin Pathol 54: 624-630, 2001

    Google Scholar 

  7. Ben-Hur H, Mor G, Inslerand V, Blickstein I, Amir-Zaltsman Y, Sharp A, Globerson A, Kohen F: Menopause is associated with a significant increase in blood monocyte number and a relative decrease in the expression of estrogen receptors in human peripheral monocytes. Am J Reprod Immunol 34: 363-369, 1995

    Google Scholar 

  8. Cao S, Hundall SD, Kohen F, Lu LJW: Measurement of estrogen receptors in intact cells. Cytometry 41: 109-114, 2000

    Google Scholar 

  9. Oxenhandler RW, McCune R, Subtelney A, Truelove C, Tyrer W: Flow cytometric determination of estrogen receptors in intact Cells. Cancer Res 44: 2516-2523, 1984

    Google Scholar 

  10. Brotherick I, Lennard TWJ, Cook S, Johnstone R, Angus B, Winthereik MP, Shenton BK: Use of the biotinylated antibody DAKO-ER 1D5 to measure estrogen receptor on cytokeratin positive cells obtained from primary breast cancer cells. Cytometry 20: 74-80, 1995

    Google Scholar 

  11. Brotherick I, Shenton BK, Lennard TWJ: Are fine needle breast aspirates representative of the underlying solid tumour-a comparison of receptors levels, ploidy and the influence of cytokeratin gates. Br J Cancer 72: 732-737, 1995

    Google Scholar 

  12. Brotherick I, Browel DA, Shenton BK, Egan M, Cunliffe, Webb LA, Lunt LG, Young JR, Higgs M: The effect of 3-week tamoxifen treatment on oestrogenreceptor levels in primary breast tumours: a flow cytometry study. Br J Cancer 77: 1657-1660, 1998

    Google Scholar 

  13. Girdler I, Browell DA, Cunlife WJ, Shenton BK, Hemming JD, Scorer P, Jung JR, Brotherick I: Use of the monoclonal antibody DAKO-ERβ (8D5-1) to measure oestrogen receptor beta in breast cancer cells. Cytometry 45: 65-72, 2001

    Google Scholar 

  14. Filippini G, Griffin S, Uhr M, Eppenberrger H, Bonilla J, Cavalli F, Soldati G: A novel flow cytometric method for the quantification of p53 gene expression. Cytometry 31: 180-186, 1998

    Google Scholar 

  15. Dragowska WH, Lopes de Menezes DE, Sartor J, Mayer LD: Quantitative flouorescence cytometric analysis of bcl-2 levels in tumor cells exhibiting a wide range of inherent bcl-2 protein expression: Correlation with western blot analysis. Cytometry 40: 346-352, 2000

    Google Scholar 

  16. Henderson LO, Marti GE, Gaigalas A, Hannon WH, Vogt Jr RF: Terminology and nomenclature for standardization in quantitative fluorescence cytometry. Cytometry 33: 97-105, 1998

    Google Scholar 

  17. Berthois Y, Katzenellenbogen JA, Katzenellenbogen BS: Phenol Red in tissue culture media is a weak estrogen: implications concerning the study of estrogen-responsive cells in culture. Proc Natl Acad Sci 83: 2496-2500, 1986

    Google Scholar 

  18. Darbre P, Yates J, Curis S, King RJB: Effect of Estradiol on human cancer cells in culture. Cancer Res 43: 349-354, 1983

    Google Scholar 

  19. Lin VC-L, Ng EH, Aw SE, Tan MG-K, Ng EH-L, Ho GH: Progestins inhibit the growth of MDA-MB-231 cells transfected with progesterone receptor complementary DNA. Clin Cancer Res 5: 393-403, 1999

    Google Scholar 

  20. Jiang SY, Jordan VC: Growth regulation of estrogen receptor negative breast cancer cells transfected with complementary DNAs for estrogen receptor: J Natl Cancer Inst 84: 580-591, 1992

    Google Scholar 

  21. Zenger VE, Vogt R, Mandy F, Schwartz A, Marti GE: Quantitative flow cytometry: inter-laboratory variation. Cytometry 33: 138-145, 1998

    Google Scholar 

  22. EORTC breast cancer co-operative group: Revision of standard for the assessment of hormone receptors in human breast cancer: report of the second EORTC workshop, held on March 16-17, 1979 in the Netherlands Cancer Institute. Eur J Cancer 16: 1513-1515, 1980

    Google Scholar 

  23. Wei LL, Kretn NL, Francis MD, Gordon DF, Wood WM, O'Malley BW, Horwitz KB: Multiple human progesterone receptor messenger ribonucleic acids and their autoregulation by progestin agonists and antagonists in breast cancer cells. Mol Endocrinol 2: 62-72, 1998

    Google Scholar 

  24. Alarid ET, Bakopoulos N, Solodin N: Proteasome-mediated proteolysis of estrogen receptor: a novel component in autologous down regulation. Mol Endocrinol 13: 1522-1534, 1999

    Google Scholar 

  25. Parl FF, Posey YF: Discrepancies of the biochemical and immunohistochemical estrogen receptor assays in breast cancer. Hum Pathol 19: 960-966, 1988

    Google Scholar 

  26. Stearns V, Singh B, Tsangaris T, Crawford JG, Novielli A, Ellis MJ, Isaacs C, Pennamen M, Tibery C, Farhad A, Slack R, Hayes DF: A prospective randomized pilot study to evaluate predictors of response in serial core biopsies to single agent neoadjuvant doxorubicin or paclitaxel for patients with locally advanced breast cancer. Clin Cancer Res 9: 124-133, 2003

    Google Scholar 

  27. Love RR, Duc NB, Havighurst TC, Mohsin SK, Zhang Q, DeMets DL, Allred DC: Her-2/neu overexpression and response to oophorectomy plus tamoxifen adjuvant therapy in estrogen receptor-positive premenopausal women with operable breast cancer. J Clin Oncol 21: 4532-4557, 2003

    Google Scholar 

  28. McCatrhy NJ, Yang X, Linnoila IR, Merino MJ, Hewitt SM, Parr AL, Paik S, Steinberg SM, Hartmann DP, Mourali N, Levine PH, Swain SM: Microvessel density, expression of estrogen receptor alpha, MIB-1, p 53, and c-erbB-2 in in-flammatory breast cancer. Clin Cancer Res 8: 3857-3862, 2002

    Google Scholar 

  29. Gratama JW, D'hautcort JL, Mandy F, Rothe G, Barnett D, Jannossy G, Papa S, Schmitz G, Lenkei R: Flow cytometric quantitation of immunofluorescence intensity: problems and perspectives. Cytometry 33: 166-178, 1998

    Google Scholar 

  30. Lenkei R, Gratama JW, Rothe G, Schmitz G, D'hautcort JL, Arekrans A, Mandy F, Marti G: Performance of calibration standards for antigen quantitation with flow cytometry. Cytometry 33: 188-196, 1998

    Google Scholar 

  31. Osborne CK, Zhao H, W Fuqua SA: Selective estrogen receptor modulators: structure function and clinical use. J Clin Oncol 18: 3172-3186, 2000

    Google Scholar 

  32. Charpin C, Martin PM, De Victor B, Lavaut MN, Habib MC, Andrac L, Toga M: Multiparametric study (SAMBA 200) of estrogen receptor immunocytochemical assay in 400 human breast carcinomas: analysis of estrogen receptor distribution heterogeneity in tissues and correlations with dextran charcoal assays and morphological data. Cancer Res 48: 1578-1586, 1988

    Google Scholar 

  33. Palmari J, Dussert C, Berthois Y, Penel C, Martin PM: Distribution of estrogen receptor heterogeneity in growing MCF-7 cells measured by quantitative microscopy. Cytometry 27: 26-35, 1997

    Google Scholar 

  34. Ricketts D, Turnbull L, Ryall G, Bakhshi R, Rawson NS, Gajet JC, Nolan C, Coombes RC: Estrogen and progesterone receptors in the normal female breast. Cancer Res 51: 1817-1822, 1991

    Google Scholar 

  35. Vogt RF, Marti GE, Schwartz A: Quantitative calibration of fluorescence intensity for clinical and research applications of immunophenotyping by flow cytometry. In: Tyrer HW (ed.) Reviews of Biotechnology and Bioengineering, Vol. 1: Cytometry, Ablex Publishing Corporation, Norwood, NJ, 1994

    Google Scholar 

  36. Katzenellenbogen BS, Kendra KL, Norman MJ, Berthois Y: Proliferation, hormonal responsiveness, and estrogen receptor content of MCF-7 human breast cancer cells grown in the short term and long term absence of estrogens. Cancer Res 47: 4335-4360, 1987

    Google Scholar 

  37. Luqmani YA, Temmim L, Memon A, Ali MAA, Parkar AH: Steroid receptor measurement in breast cancers: comparison between ligand binding assay and enzyme-immunoassay in cytosolic and nuclear extracts. Int J Cancer 71: 526-538, 1997

    Google Scholar 

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Authors and Affiliations

  1. Department of Immunology, Saint Savas Cancer Hospital, Athens, Greece

    Angelos D. Gritzapis, Constantin N. Baxevanis & Michael Papamichail

  2. Breast Cancer Clinic, Saint Savas Cancer Hospital, Athens, Greece

    Ioannis Missitzis

  3. Molecular Endocrinology Programme, Institute of Biological Research and Biotechnology, the National Hellenic Research Foundation, Athens, Greece

    Efrosine S. Katsanou & Michael N. Alexis

  4. Hormone Receptor Unit, Saint Savas Cancer Hospital, Athens, Greece

    Julia Yotis

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  1. Angelos D. Gritzapis
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Gritzapis, A., Baxevanis, C., Missitzis, I. et al. Quantitative Fluorescence Cytometric Measurement of Estrogen and Progesterone Receptors: Correlation with the Hormone Binding Assay. Breast Cancer Res Treat 80, 1–13 (2003). https://doi.org/10.1023/A:1024462416640

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