From The Editors  What's New At MEDICC  Spotlight On...  MR Interviews…  Health News from Cuba  Cuban Medical Research

CUBAN MEDICAL RESEARCH

Presence of estrogen receptor and Her2 in a sample of breast cancer patients
Rosa Irene Alvarez Goyanes 1,
Xiomara Escobar Pérez 2 ,
Rolando Camacho Rodríguez 3,
Leticia Llanes Fernández 4,
Maybi Orozco López 5,
Julia Cruz Mojarrieta 6,
Hilda María Rodríguez Montero 7,
Maria Emilia Juliá 8.
National Institute of Oncology and Radiology. Havana City. Cuba.

Introduction

In the treatment of cancer, the use of tumor markers with prognosis and predictive value in response to cancer drugs such as the Estrogen Receptor (ER) enables a more selective and effective therapy. The estrogen receptor has prognosis value and can influence therapy decisions in the case of patients with breast cancer 1,2,3. The Presence of ER in tumor tissue is not only indicative of better prognosis of the patient’s condition, or in other words, longer event-free survival as well as an increase in longevity . Besides, patients with positive ER have a higher probability of positive response to hormone therapy 4-10

In Cuba, ER was first given in 1972 by means of biochemical methods such as dextran-coated charcoal followed by Scatchard plotting 11,12, the isoelectric approach method 13,14,15 and the sucrose-density gradient. This technology was used for almost two decades iat the Institute of Oncology and Radiobiology in Havana City and at the Santiago de Cuba Oncology Hospital.

This method has many disadvantages such as: the use of radioactive hormones, lots of sample to process, expensive equipment and, in some cases, damage to the structure of the receptor 16.

In the 1980´s immunohistochemical methods (IHC) for ER detection 17-19 were developed. These methods recognize the receptor through the use of monoclonal antibodies and provide fast and high sensitivity diagnosis.

At present another marker with possible prognosis value and especially predictive value for breast cancer is the Her2 gene expression. It may be amplified and its protein receptor over expressed in more than 20% of breast tumors. This indicates the gene can play an important role in cell malignancy 20.

It is generally accepted that over expression of Her2 is associated with aggressive growth and a worse prognosis in invasive breast cancer, especially in the case of metastatic lymph nodes 21. Clinically, over expression and/or amplification of Her2 in breast cancer is associated with negative ER tumors, anomalies of p53, negative bcl-2, high mitosis values and lymph node infiltration, reduced response to chemotherapy and hormone therapy 20. However, it has not been possible to come to definitive conclusions about impact on Her2 in response to endocrine therapy and chemotherapy.

Positive Her2 is indicative of a reduced response to hormone therapy, especially therapy with tamoxifen. The relationship between Her2 status and chemotherapy is weaker and less defined. Some studies suggest that a positive Her2 is associated with particular sensitivity to antraciclins and resistance to CMF therapy. Other studies show contradictory results. Her2 as a tumor marker with predictive response value is applicable in target treatments such as Trastuzumab (Herceptin ) 22.

In practice, IHC is the most frequently used method for Her2 status determination. With this method it is possible to individually identify over expression of protein Her2 in tumor cells 21.

Material and Methods

The expression of both markers in a sample of breast cancer patients was determined.

ER determination was done in 412 breast cancer patients coming from hospitals in five provinces of Cuba. The study included the use of tissue fragments fixed in formalin and embedded in paraffin of the Pathological Department. IHC technique was used. The monoclonal antibody against ER, a multilink kit as a detection system for streptavidin – Biotin- Peroxidase (ultra sensitive) and a development color kit of 3,3 ’ diaminobencidine Tetrahydrochloride ( DAB ) employed was from Anacrom Diagnosis, S.L.

In each case two sections (4 um)of routine fomalin fixed, paraffin embedded blocks were cut and mounted on silanized slides and were incubated at 68 oC for 12 hours..

The sections were deparaffinized in xylene, hydrated in decreasing concentrations of ethanol,cleared for five minutes in distilled H 2O and washed with buffer tris saline(TBS) Antigenic exposure of ER was done by boiling the plates in citrate buffer pH 6,8 for 10 minutes in a microwave oven. The slides cooled for 20 minutes, were washed in distilled water and later in TBS solution . After endogen tissue peroxidase activity was inhibited using a 3% H 2O 2 solution for 5 minutes at room temperature (RT) and washed with TBS. Sections were incubated with the anti-ER monoclonal antibody and TBS as negative control for 30 minutes at room temperature. After washing, a biotinylated antibody was used for 10 minutes, followed by streptavidin- peroxidase complex applied for the same period of time and at RT. Finally the peroxidase reaction was developed using diaminobenzidine ( DAB ) solution as chromogen. Sections were counterstained with mayer hematoxylin follwewd by dehydration in increasing ethanol concentration, cleared in xylene and mounted in Eukitt(Kinder GmbH & Co).

Each of the experiments used a positive ER tissue as control.

Evaluation was carried out in a semi quantitative way by means of a light microscope (Leica ).

For Her2 determination, a total of 85 patients with ductal infiltrating mammary carcinoma were studied. These patients were registered at the National Institute of Oncology and Radiobiology. For this study routine tissue blocks fixed in formalin and embedded in paraffin from the Pathology Department were used. These include cases from 1996 until 2003.

Determination of the over expressed protein was done by means of IHC technique, using the diagnosis kit for Her2 determination, DAKO Hercep Test TM.

IHC technique was implemented as described previously for the ER. Some variations occurred such as: the unmasking time for Her2 receptor was 40 min; anti- Her2 antibody and negative control were applied for 30 minutes at room temperature; visualization reagent was used for the same period of time and at RT, and the tissue sections were incubated with DAB for 10 minutes.

A control slide from DAKO Hercep Test TM was used in each experiment. It has three cellular clones with different Her2 oncoprotein expression.. Alternative controls were also used.

Evaluation was carried out in a semi quantitative way by means of a light microscope (Leica) and the score follows the classification proposed by DAKO Hercep test user guide and accepted by FDA:

Negative ( 0 ) , absence of staining in all or in part of the cytoplasm membrane <10% of the tumor cells.

Negative ( +1 ) , faint staining in part of the cytoplasm membrane > 10% of the tumor cells.

Slightly positive ( +2 ),weak or moderate staining in all the cytoplasm membrane in > 10% of the tumor cells.

Strongly positive ( +3 ), strong staining in all the cytoplasm membrane in > 10% of the tumor cells.

Results and Discussion

Estrogen Receptor

In 1988 in the Oncology Institute, Pascual M.R., et al 23 studied ER expression in 403 breast cancer patients with biochemical methods specifically dextran coated charcoal techniques. They found 54% ER+ patients and 43% ER-. In our study 412 patients were included; 182 were ER+ which accounts for 44,17%, and 230 were ER- which accounts for 55,82%.

Many studies have shown a strong correlation between both methods 24,25,26. There are differences in the results of the two methods. As to the characteristics of the samples used, the first method used fresh tissues while the IHC method used fragments of tissue fixed in formalin and embedded in paraffin. As to the way of quantifying ER, it is quantitative for one method through maceration of the tumor fragments, which brings about loss of cell integrity; for the other method, quantifying is semi quantitative through direct visualization of nuclear staining of the cells in tissue sections. These differences account for the fact that in our study positive percentages of ER are slightly lower.

In our case, the conditions under which the samples were fixed and processed in different hospitals, the differences as to the quality of the samples processed in different years in which they were collected and the fact that the samples were kept and processed without considering the possibility of IHC determinations, could have an influence on antigen conservation. Antigen loss might occur and thus an increased number of false negatives could result.

Fig 1. Examination of AcM anti ER, nuclear marking

Her2.

As shown in table I., the 85 samples studied display all the score pattern reported for this protein. This is shown in figure 2. (A, B, C. )

Table.1. Her2 detection by means of Immunohistochemistry.

Fig 2.A Examination of AcM anti Her2, marking 1+

Of the 85 cases studied, 57 were negative with 0 score. They showed total absence of staining in all or part of the membrane in less than 10% of the cells. Another group of six patients was classified as negative too but the score was 1+ because a fine stain was found in part or in all the membrane in more than 10% of the cells.

Fig 2.B Examination of AcM anti Her2, marking 2+

Of the positive groups, 6 samples were classified as slightly positive with a score of 2+ due to weak or moderate staining of all the cytoplasm membrane. Sixteen samples were strongly positive(3+) with strong staining of the cytoplasm membrane. In both groups more than 10% of the cells were analyzed.

When considering the percentages in each of the 4 groups, we found that the largest percentage corresponds to the negative cases which were 74,1%.

Fig 2.C Examination of AcM anti Her2, marking 3+

In the group classified as positive, 7.05% were slightly positive and 18,82% were 3+ or strongly positive. The total percentage of this group was 25,87%. This percentage is in the range reported by literature which goes from 20 to 30% of overexpression of this oncoprotein. protein 27,28,29

It is interesting to underline that only in patients with a score of 3+ , a high level of response to treatment with Trastuzumab is expected 30.

According to our results, negative cases accounted fort the largest percentage (74,55%), followed by the cases with the highest intensity score(18,82%)

BIBLIOGRAPHY

1. Mc Guire WL, Clark GM, Dressler LG, Owens MA. Role of steroid hormone receptors as pronostic factors in primary breast cancer. NCI Monographs. 1:19-23, 1986

2. Rose C, Thorpe SM, Andersen KW, et al. Beneficial effect of adjuvant tamoxifen therapy in primary breast cancer patients with high estrogen receptor values. Lancet ; 1:16-9, 1985

3. Stewart HJ, Prescott R. Adjuvant tamoxifen therapy and receptors levels. Lancet. 1:573, 1985

4. Pascual M.R. Determinación de receptores hormonales de tumores mamarios humanos. Rev Cub Obstet Ginecol 7:1, 1981.

5. Lage A. et al. Factors associated with prognosis in human breast cancer: predictors for evolution and relapse. Neoplasm 30:345, 1983.

6. Macías A. et al. Factors associated with prognosis in human breast cancer: progesterone receptors and clinical factors. Neoplasm 30:, 1983.

7. Pascual M.R. et al. Factors associated with prognosis in human breast cancer.II. Multivariate stratification analysis. Neoplasm 30:485, 1983.

8. Pascual M.R. et al. Factors associated with prognosis in human breast cancer.III. Estradiol receptor and short term relapse. Neoplasm 30:589, 1983.

9. Pascual M.R. et al. Factors associated with prognosis in human breast cancer. V. The simultaneous use of estrogen and progesterone receptor measurements for prediction of short-term relapse. Neoplasm 32:247, 1984.

10. Reiner A. et al. Immunocytochemical localization of estrogen and progesterone receptor and prognosis in human primary breast cancer. Cancer Res 50:7057, 1990.

11. Scatchard G: The attractions of proteins for small molecules and ions. Ann NY Acad Sci 51:660-672, 1949

12. Chamness GC and McGuire WL: Scatchard plots: common errors in correction and interpretation. Steroids 26(4): 538-542, 1975

13.Wrange Ö, Nordenskjöld B, Silfverswärd C, Granberg PO and Gustafsson J-A: isoelectric focusing of estradiol receptor protein from human mammary carcinoma - a comparison to sucrose gradient analysis. European Journal of cancer 12: 695-700, 1976.

14.Wrange Ö, Nordenskjöld B and Gustafsson J-A. Cytosol estradiol receptor in human mammary carcinoma: an assay based on isoelectric focusing in polyacrylamide gel. Analytical Biochemistry 85: 461-475, 1978.

15.Wrange Ö, Humla S, Ramberg I, Gustafsson S, Skoog L, Nordenskjöld B and Gustafsson J-A: Progestin-receptor analysis in human breast cancer cytosol by isoelectric focusing in slabs of polyacrilamide gel. Journal of Steroid Biochemistry 14: 141-148, 1981.

16. .Mårten Ferno, Åke Borg and Allan Norgren. A comparison of two steroid receptor assays in breast cancer: Dextran coated charcoal and isoelectric focusing. Anticancer Research. 3:243-246, 1983.

17. Jensen E.V. et al. Receptors reconsidered: a 20 year perspective. Rec Prog Horm Res 38:1, 1982.

18.Weigand R.A. et al. Quantification of progesterone receptor in human breast tumors by double monoclonal enzyme immunoassay. Breast Cancer Res Treatment 8:87, 1986.

19. Greene G. and Press M.F. “Immunochemical Evaluation of Estrogen Receptor and Progesterone Receptor in Breast Cancer” En: Immunological Approaches to the Diagnosis and Therapy of Breast Cancer. Ed. R Cerani. Plenum Publishing Corp.; New York, pp. 119-135, 1987.

20. Dawen X, Xiao- Ou S, Zonglin D, Wan-Quing W, Kim E. C, Qi D, Yu- tang G, Fan J, Wei Z. Population – based, case- control study of Her/2 genetic polymorphism and breast cancer risk. Journal of the National Can. Inst.92; 5: 412- 417, 2000.

21. van de Vijver M. J. Assessment of the need and appropriate method for testing for the human epidermal growth factor receptor- 2 (Her/2). European Journal of cancer. 37: S11-S17, 2001.

22. Piccart M, Lohrisch C, Di Leo A, Larsimont D. The predictive value of Her/2 in breast cancer. Oncology 61; (supp 2): 73-82, 2001.

23. Pascual M.R. Factores clínicos y bioquímicos en el pronóstico del cáncer de mama. Tesis para optar por el grado de Doctor en Ciencias. INOR, sept. 1988.

24. King W. J, DeSombre E.R, Jensen E. D, Greene J. L. comparison of immunocytochemical and steroid – binding assays for estrogen receptor in human breast tumors. Cancer Res 1985; 45:293- 304.

25. Pertschuk L. P, Einsenberg K. B, Carter A.C, Feldman J.G. Immunohistologic localization of estrogen receptors in breast cancer with monoclonal antibodies. Correlation with biochemistry and clinical endocrine response. Cancer 1985; 55:1513- 1518.

26. Sondegaard G, Pedersen K.O , Paulsen S.M. Estrogen receptor analyses in breast cancer: comparison of monoclonal immunohistochemical and biochemical methods . Eur. J. Cancer Clin. Oncol 1989; 25: 1425- 1429.

27. Esteva- Lorenzo F.J, Sastry L, King C.R. The erb B- 2 gene: From research to application, in Dickson R., Salomon D.S ( eds ): Hormones and Growth factors in Development and Neoplasia. New York, N.Y, 1998, pp 421- 444.

28. Slamon D.J, Clark G.M,Wong S,G, Levin W,J,Ullrich A, Mcguire W.L. Human breast cancer: correlation of relapse and survival with amplification of the Her2-2/neu oncogene. Science 1987: 235:177- 182.

29. Salmón D.J, GodolphinW, Jones L.A, , et al. Studies of the Her-2/neu proto oncogene in human breast and ovarian cancer. Science 1989: 244: 707- 712.

30. Slamon D.J, Leyland- Jones B, Shak S, Fuchs H, Paton V, Bajamonde A, Fleming T, Eiermann W, Wolter J, Pegram M, Balsega J, Norton L. Use of chemotherapy plus a monoclonal antibody against her2 for metastatic breast cancer that overexpresses Her2. The New England Journal of Medicine 2001: 344:783- 792.

1- Biology Degree. Laboratory of experimental Immunohistochemistry

2-Degree in Health Technology. Laboratory of experimental Immunohistochemistry

3- 2 nd Degree Specialist in Oncology. Mastopathy Service

4- Biochemistry Degree. Laboratory of Immunology.

5-Pathologic Anatomy Technician. Laboratory of experimental Immunohisto chemistry

6- 2nd Degree Specialist in Pathologic Anatomy, Doctor in Sciences. Pathologic Anatomy Laboratory

7- Biology Degree. Laboratory of experimental Immunohistochemistry

8- Pathologic Anatomy Technician. Pathologic Anatomy Laboratory