An immunohistochemical assay for the measurement of estrogen receptor (ER) has been evaluated on 290 consecutive human breast biopsy and mastectomy specimens in the year 1992 at The Aga Khan University Hospital laboratories. Immunohistochemical localization of estrogen receptor on frozen/paraffin section was scored in a semi-quantitative fashion incorporating both the intensity and the distribution of specific staining. Histologic grading of the tumour was performed according to Bloom’s method. In this study, 21% of the tumours were estrogen receptor negative, 15% were weak positive, 25% intermediate positive and 39% strong positive. Fifty percent of the well differentiated tumours showed strong ER positivity against 27% of the poorly differentiated tumours. Seventy eight percent of all negative estrogen receptors were in patients younger than 50 years of age (pre-menopausal group), while 52% of strong estrogen receptor positivity was observed in patients older than 50 years (post- menopausal). This study demonstrates the value of immunohistochemical method to determine the ER status in patients with advanced breast cancer

Aijaz, F.

Aziz, S. A.

Hasan, Sheema H.

Naqvi, M.

Pervez, Shahid

Shaikh, S.

eCommons@AKU

eCommons@AKUDepartment of Pathology and Laboratory Medicine Medical College, PakistanJune 1994Immunohistochemical Estrogen receptordetermination in human Breast carcinoma:correlation with histologic differentiation and age ofthe patientsShahid PervezAga Khan University, shahid.pervez@aku.eduS. ShaikhAga Khan UniversityF. AijazAga Khan UniversityS. A. AzizAga Khan UniversityM. NaqviAga Khan UniversitySee next page for additional authorsFollow this and additional works at: http://ecommons.aku.edu/pakistan_fhs_mc_pathol_microbiolPart of the Oncology Commons, Pathological Conditions, Signs and Symptoms Commons,Pathology Commons, and the Women's Health CommonsRecommended CitationPervez, S., Shaikh, S., Aijaz, F., Aziz, S. A., Naqvi, M., Hasan, S. H. (1994). Immunohistochemical Estrogen receptor determination inhuman Breast carcinoma: correlation with histologic differentiation and age of the patients. Journal of Pakistan Medical Association,44(6), 133-136.Available at: http://ecommons.aku.edu/pakistan_fhs_mc_pathol_microbiol/312AuthorsShahid Pervez, S. Shaikh, F. Aijaz, S. A. Aziz, M. Naqvi, and Sheema H. HasanThis article is available at eCommons@AKU: http://ecommons.aku.edu/pakistan_fhs_mc_pathol_microbiol/312Immunohistochernical Estrogen Receptor Determination inHuman Breast Carcinoma: Correlation with HistologicDifferentiation and Age of the PatientsPages with reference to book, From 133 To 136 S. Pervez, H. Shaikh, F. Aijaz, S.A. Aziz, M. Naqvi, Sheema H. Hasan ( Department of Pathology, The Aga KhanUniversity Hospital, Stadium Road, Karachi. ) Abstract An immunohistochemical assay for the measurement of estrogen receptor (ER) has been evaluated on290 consecutive human breast biopsy and mastectomy specimens in the year 1992 at The Aga KhanUniversity Hospital laboratories. Immunohistochemical localization of estrogen receptor onfrozen/paraffin section was scored in a semi-quantitative fashion incorporating both the intensity andthe distribution of specific staining. Histologic grading of the tumour was performed according toBloom’s method. In this study, 21% of the tumours were estrogen receptor negative, 15% were weakpositive, 25% intermediate positive and 39% strong positive. Fifty percent of the well differentiatedtumours showed strong ER positivity against 27% of the poorly differentiated tumours. Seventy eightpercent of all negative estrogen receptors were in patients younger than 50 years of age (pre-menopausal group), while 52% of strong estrogen receptor positivity was observed in patients olderthan 50 years (post- menopausal). This study demonstrates the value of immunohistochemical methodto determine the ER status in patients with advanced breast cancer (JPMA 44:133, 1994).Introduction The clinical significance of estrogen receptor assays is well recognized. There is a general agreementthat approximately half of the women whose tumours have detectable estrogen receptor (ER) willobtain objective remission from some form of endocrine therapy1. This number increases to threequarters when progesterone receptor (PR), an estrogen induced protein is included1. Besides this, asignificantly prolonged overall survival among patients with ER-positive tumours has been reported2.In these patients, disease free interval (DPI) is prolonged not because ER-positive patients respondedbetter to adjuvant hormonal treatment, but because ER-positive and ER-negative tumours havedifferent biological behaviour. Knowledge of estrogen receptor status is therefore, of both therapeuticand prognostic importance. There are numerous theoretical and practical advantages for thedemonstration of estrogen and progesterone receptors by immunohistochemical (IHC) means overcytosol based biochemical assays3,4. Breast cancers are frequently heterogenous bothmorphologicallyand in terms of biological behaviour. This is also true for estrogen receptor5. A majordisadvantage of tissue homogenates used for biochemical ER measurements is that it cannot assessintratumoural heterogeneity. The development of monoclonal antibodies against the ER proteins havebeen shown to be aplicable to the demonstration of ER by IHC method6 . This technique offers anadvantage of a reproducible, easy to interpret staining product which may be scored in asemiquantitative fashion. This study documents the value of 133 this method to predict the prognosisand response to hormonal treatment in patients with advanced breast cancer.Materials and Method Patient PopulationThis study group consisted of 290 consecutive human breast biopsy and mastectomy specimensreceived for ER evaluation in the year 1992 at The Aga Khan University Hospital, Department ofPathology, Karachi. Age of the patients ranged from 22-85 years (median 46 years). The most commontype of tumour was infiltrating duct carcinoma (Table I).Immunohistochemical proceduresPrimary antibodies: Monoclonal antibody directed against human ER protein (rat monoclonal, AbbottLaboratories) was used at recommended dilution on P.S. In some cases where the tumour was alreadyfixed in formalin, anti-estradiol antibody (Signet Rabbit AB Laboratories Inc.) was used. 3-5 um thickfrozen sections were cut and mounted on gelatin coated slides. The slides were immediately immersedin 4% formaldehyde-phospha.te-buffered saline for 10 minutes at room temperature. This was followedby 3-5 minutes immersion in 100% methanol and 1-3 minutes in acetone at -4°C. For paraffinembedded tumours, 3 um sections were cut and mounted on coated slides. Sections were stained by 3step PAP (peroxidase anti- peroxidase) technique. Positive and negative controls were included witheach panel.Histological grading of tumourHistological grading of the tumours (infiltrating ductal carcinomas) was performed according to themethod described by Bloom and Richardson7 taldng into consideration 3 criteria, i.e., tubule formation,nuclear pleomorphism and mitosis, each given a point range from 1-3. Grade is allocated as follows: 3-5 points (grade 1, well differentiated, 6-7 points (grade II, moderately differentiated) and 8-9 points(poorly differentiated).Semi-quantitative evaluation of immunocytochemical stainingThe immunohistochemical localization of ER was scored in a semi- quantitative fashion incorporatingboth the intensity and the distribution of specific staining as described by Kenneth S. McCarty, Jr. et al.6,8. The evaluations were recorded as percenntages of positively stained tumour cells in each of fiveintensity categories denoted as 0 (no staining),. 1 + (weak but detec table, 2 + (mildly distinct), 3 +(moderately distinct) and 4 + (strong). For each tissue, a value designated as HSCORE was derived bysumming the percentages of cells staining at each intensity multiplied by the weighted intensity ofstaining. An HSCORE of <74 was established as negative, between 75-99 as weak positive, 100-119 asintermediate positive, while 120 and more as strong positive. An example from one of the case reportsis given below:Results i) Patterns of stainingImmunohistochemical evaluation using monoclonal antibody to estrogen receptor protein onfresh/frozen tumour sections revealed specific staining localized to nuclei of target cells only (Figure1).No cytoplasmic staining was seen. In contrast two different staining patterns were observed in formalinfixed/paraffin embedded tumour sections using anti-estradiol antibody. The first pattern consisted ofpositive reaction products within the cytoplasmin most carcinoma cells. The second patterndemonstrated positive reaction product both in the nucleus and cytoplasm. No staining was observed inparaffin embedded tissues using anti-ER monoclonal antibody even with trypsin digestion.ii) Histologic grade and estrogen receptor statusIn this study, 14% of the tumours (infiltrative duct carcinoma) were graded as grade 1 (welldifferentiated), 48% as grade 2 (moderately differentiated) and 38% as grade 3 (poorly differentiated).Twenty-one percent (21%) of the total tumours showed negative estrogen receptor status. In positivegroup, 15% of the tumours were weak positive, 25% intermediate positive and 39% strong positiye forER 50% of the well differentiated tumours showed it rong ER positivity against 27% of the poorlydifferentiated tumours in positive group (Table II).By using Chi-square statistical analysis (grade 1 versus grade 3), results are statistically significant, i.e.,Pc 0.05 (0.023).iii) Patient age and estrogen receptor statusA significant correlation also exists between ER status and age at the time of diagnosis with 78% of allnegative estrogen receptors reported in patients younger than 50 years of age (pre- menopausal group).Fifty-two percent (52%) of strong estrogen receptor positivity was reported in patients older than 50years (post-menopausal group).Discussion A number of histochemical techniques have provided ways to visualize ER in tissue sections2-4,6,8.These approaches are underlying intense study to determine if they can improve the predictive accuracyof biochemical assays for ER. Several studies correlating the biochemical and immunohistochemicalmethods appeared in literature indicates that a high degree of specificity and sensitivity for ER can beattained by IHC approach with MAbs to ER6,9. At the present time, one indisputable application of IHCapproach is the very small neoplasm in which the amount of tumour available is inadequate for cytosol-based biochemical methods. Another area of practical use of this method is when a small carcinoma isdetected only after the tissue has been processed and embedded in paraffin2,10. One drawback of IHCover biochemical method is that the former is at best semi-quantitative. However, several methods arebeing employed to quantitate the results of IHC for ER in attempts to facilitate comparison with thecytosoltechnique. These range from estimating the percentage of neoplastic cell nuclei immunostainedto the use of computerized cell analyses system. There is no doubt, however, that the histochemicalresults have enlarged the pathologists’ knowledge of ER distribution with in the tumour. Some tumoursare composed of both ER positive and ER- negative cells. Observations about heterogeneity of stainingenables to improve predictive accuracy in patients whose ER positive tumours may contain significantpopulation of hormone unresponsive cells. A certain number of false positive and false negative resultsmay occur. Anti-factual heterogeneity of immunostaining is not unconunon informahn flxedtissues. Insome cases this may pose problems. Although most positive cases are easily discernible, there areoccasional examples of ambiguous results which may benefit from a system which more clearly definesa quantitative threshold more particularly if backed up by clinical response data. The false positives areviewed with somewhat less concern than the false negatives. This is because the modem hormonaltherapy, e.g., tamoxifen is relatively free of side effects and the lack of response to therapy becomesapparent within a few weeks, allowing the clinician to move onto other modalities. The frequency offalse positive results may be reduced if hormonal treatment can be reserved for the sub-set of patientswith tumours containing both estrogen and progesterone receptors. Evidence from animalmodelssuggests that PR is under the control of ER. Presence of PR indicates that the ER mechanismis not onlypresent but also functional. Because ER-ICA detects ER by its antigenicity, it does not assure than thereceptors are functional. False negative results represent patients who could have received tangiblebenefits from benign therapy. Sampling and handling errors are important reasons for false negativity,while analytical errors can be minimized by performing parallel assays on control material, It has beenfound that high levels of plasma estrogen may induce false negative results11 because the endogenousestrogen occupies the binding receptor protein. This effect might be diminished by performing biopsiesduring menses. Biochemical methods are also not foolproof and on occasion ER positivity may in factbe an artefact because of ER presence in adjacent benign tissue. This technique, using anti-ERmonoclonal antibodies on fresh/frozen tumour sectiona demonstrates nuclear localization of the type 1,high affinity receptor without convincing evidence for cytoplasmic receptor staining. This findingcontrasts with cytoplasmic localization in histochemical methods employing anti- estrogen (anti-estracliol) antibodies. These antibodies probably detect type II and III estrogen binding sites rather thanthe type I high affinity receptor12. Inspite of these problems/criticisms there have been several reportsof a good agreement between qualitative results obtained by histochemical method using anti-estradiolantibody and biochemical methods13,14 . This, probably represents/supports the theory of two stepinteraction mechanism of the receptor protein. Namely, the steroid hormone first binds with thecytoplasmic receptor protein to form estrogen receptor complex and with subsequent transformation ofthe receptor protein, the complex translocates into the nucleus to bind with the nuclear chromatin15. Asignificantly prolonged overall survival among patients with ER-positive tumours has beenreported9,16,17. Disease free interval (DPI) is prolonged in these patients not because ER- positivepatients responded better to adjuvant hormonal treatment but because ER positive and ER negativetumours have different biologic properties, e.g., proliferation rate (study in progress in our departmentusing Ki-67 MAb). Several studies assessing proliferation index have shown an association betweenER content and tumour differentiation18-20. Meyer et al.18 found a significant correlation between thelow thymidine labelling index and increased ER levels in breast cancer cell. Highly proliferative,poorly differentiated tumour cells showed low ER levels. These observations have been confirmed bySilva et al20 who also found consecutive decrements of ER and PR levels as measure of differentiationof tumour with grade I (well differentiated) having the highest and grade III (poorly differentiated)having the lowest ER and PR levels. The results in our study also indicate a correlation between thelevel of ER and the degree of differentiation. However, the number of well differentiated tumours inthis study represented approximately 1/8th of the total cases and therefore, may not be trulyrepresentative. This paper extends the observation that the ER-ICA status relates to patient’s age. In thecurrent study, high levels of ER expression were most often observed in patients more than 50 years ofage, a result in keeping with the known influence of age on receptor levels21,22 - The low level of ER inpre-menopausal patients maybe explained by the higher levels of circulating estrogens with decreasedsynthesis of ER protein as a result of negative feedback. A major outcome of this study is that about55% of the population on which ER was evaluated was under 50 years of age. This is in strikingcontrast to western studies where majority population belonged to elderly group with median age of 67years2 in one study. The data presented in this prospective study show that immunohistochemicallocalization of ER provides valuable information complementary to that obtained with standardbiochemical assays.Acknowledgement We are extremely grateful to Mrs. Amina Anwerali for her secretarial help.References 1. deSombre, E.R. Steroid receptors in breast cancer. In R.W. McDivitt, H.A. Obermsn,L Ozello and N.Kaufman, eds. The breast. Baltimore, Williams and Wilkins, pp. 149-174.2. Anderson,J. and Poulsen, H.S. Immunohistochemical oestrogens receptor determination in paraffin-embedded tissne. Prediction of responsc to hormonal treatment in advanced breast cancer. Cancer,1989; 64:1901-8.3. Esteban, J.M., Ksndslaft, P.L, Mehta, P., et aL Improvement of the quantification of oestrogen andprogesterone receptors in paraffin embedded tumour by image analysis. Am.J.Clin.Pathol., 1993;99:32-8.4. King, Wi., DeSombre, ER., Jensen, By., et sl. Comparison of immunocytochemical and steroid-binding assays for oestrogen receptor in human breast tumours. Cancer Res., 1985;45:293-304.5. Davis, B.W., Zsvs, DL, Locher, OW., et al. Receptor heterogeneity of human breast cancer asmeasured by multiple intratumoural assays of oestrogen and progesterone receptor. Bur.J.CancerClin.Oneol., 1984;20-375-82.6. McCarty, KS., Miller, US., Edwin, B.C., et al. Oestrogen receptor analyses. Correlation ofbochemiesl and immunohistochemical methods using monoelonsi anti-receptor sntibodics. Arch.Pathol. Lab. Med., 1985;109:716-21.7. Bloom, H.J.O. and Richardson, W.W. Histological grading and prognosis in breast esneer. Br.J.Cancer, 1957;11:359-77.8. McCarty, KS., Szsbo, B., Flowers, J.L, etal. Use ofamonoelonal anti-estrogen receptor antibody inthe immunohistochemical evslustion of human tumours. Cancer Res. (Suppt), 1986;46:4244S-48S.9. Teach, M., Shswwa, A. and Henderson, R. lmmunohistocbemicsl determination of oestrogen andprogesterone receptor status in breast cancer Am.J.Clin.Psthol., 1993;99:8-12.10. Shintsku, P. and Said, J.W. Detection of oestrogen receptors with monoelonal antibodies inroutinely processed formslin-fixed paraffin sections of breast carcinoma, use of D Nsse pretreatment toenhance sensitivity of the reaction. Am.J.Clin.Pathol., 1987;87:161-67.11. Oxley. D.K. Steroid receptor assays in breast cancer. Vol. 1. New Jersey, CPEC., 1982, pp. 1-7.12. Walker, R.A. The loeslizstion of receptors and binding sites with reference to steroids. Inimmunocytoehemistry, modern methods and applications. 2nd ed. Van Noorden, Julia M. Polak andSusan; 1986.13. Walker, R.A, Cove, D.H. snd Howell, A. I-Iistologiesl detection ofoestrogen reeeptorin humanbresstcareinoma. Lsncet, 1980;1:171-73.14. Pertsehuk, L.P., Gaetjen, D.O.E.. Carter, A.C., et sI. An improved histoehemical method fordetection of oestrogen receptors in mammary cancer. Am.J.Clin.Pathol., 1979;71:504-8.15. Jensen, E.V., Suzuki, T., Kawsshims, T., et aI. A two-step mechanism for the interaction of estradiolwith rat uterus. Proc. Natl. Aced. Sri. USA., 1968;59:632-38.16. Thorpe, SM., Rose, C., Rasmussen, B.B., et si. Steroid hormone receptors as prognostic indicatorsin primary breast cancer. Breast Csneer Res. Treat, 1986; 7 (Suppl):91-8.17. DeSombre, ER., Thorpe, SM., Rose, C., et al. Prognostic usefulness of oestrogen receptorimmunocytochemical assays for human breast cancer. Cancer Res., 1986;40: (Suppt) 4256S-64S.18. Meyer, 3.5., Rao, B.R., Stevens, S.C. and White, W.L., Low incidence of oestrogen receptor inbresst carcinomas with rapid rates of cellular replication. Cancer, 1977;40:2290-98.19. Mohammed, R.H., Lskstva, Di., Haus, B., et al. Oestrogen and progesterone receptors in humanbreast canceL-: correlation with histologic subtype and degree of differentis¬ tion. Csneer,1986;58:1076-81.20. Silvs, J.S., Cox, CE., Well, S.A., et st Biochemical correlates of morphologic differentiation inhuman bresstcsneer. Surgery, 1982;92:443-49.21. Terenius, U, Johansson, H., Rimsten, A., etat Malignant and benign human mammsry disease:oestrogen binding in relation to clinical dats. Cancer, 1974;33:1364-68.22. Oriffiths, K, Nicholson, Ri., Bismey, RW., et al. Oestrogen receptor determinations: studies inrelation to rapidly progressive carcinoma of the breast In: V.C. Jordan (ed).,strogen/anti-oestrogenaction and breast cancer therapy. W.L Madison, The University of Wisconsin Press, 1986,pp.325-40.

Immunohistochemical Estrogen receptor determination in human Breast carcinoma: correlation with histologic differentiation and age of the patients

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Immunohistochemical Estrogen receptor determination in human Breast carcinoma: correlation with histologic differentiation and age of the patients

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