Hormonal receptors in mammary carcinoma : comparison between quantitative and qualitative methods

Alternatives to the traditional hormone receptor dosages for prognostic evaluation and clinicai approach to breast cancer have been proposed for immunohistochemical determinations. For correlation purposes, such procedures were compared in 37 patients presenting 5 to 15 years of survival. Considering 30 fm/mg as the positivity index, the disagreement between both methods reached 35.1 % with estrogen and 48.5% with progesterone receptors. When the positiveness levei was changed to 20 fm/mg, the discrepancies were reduced to 32% with ER and increased to 57% with PgR. This study leads us to not recommend the immunohistochemical method applied to paraffin sections as an alternative procedure to the dextran-charcoal dosage for prognosis and therapeutic management of mammary carcinoma.


INTRODUCTION
T he hormonal dependence ofbreast cancer has been known and discussed for over 100 years.In 1889, Schinzinger I in Germany, was the first to call attention to this ~act and Beatson 2 in Scotland, in 1896, presented a paper showing cases of mammary carcinoma remissions after bilateral oophorectomy.
However his results did not exceed 30% of patients.After the laboratory isolation of cortisone, hormonal ablative surgery to control mammary cancer has been extended to also include adrenalectomy and hypophysectomy.
Meanwhile, endocrine additive treatments were started and successively estrogens, androgens,

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Antonio Franco.Montoro Praça Amadeu Amaral, 47 São Paulo/SP -Brasil-CEP 01327-010 progesterone, anti-hormones like tamoxifen, aromatase inhibitors, and recently LH-RH analogs, were used in order to control breast cancer.All these attempts had in common one question: to what percentage would the patients respond to this hormone manipulation?Without a selective criterion the results were similar to those show,n by Beatson, ie only one third were responsive.
By 1970 there were clinicaI and biochemical parameters to predict therapeutic results that would allow the drawing of conclusions regarding different grades of hormonal dependence.Among them, the disease-free interval proved to be worthwhile.When the time elapsed between the curative mastectomy and the frrst recurrence of disease was longer than five years, the responsiveness rate' to endocrine manipulation increased to two thirds of the patients.
In the early seventies, Jensen 3 identified the estrogen receptor (ER) in the mammary tumor cell as a reliable parameter to evaluate the grade of hormone dependence right at the beginning of treatment, without having to wait for five years of evolution ., J ensen believed that the mammary carcinoma hormonal dependence was positive when the biochemical dosages indicated a receptor amount above three fentomoles/milligram of neoplastic tissue.Lately, another "estrogenic derived marker has been identified, the progesterone receptor (PgR).Its positivity index was initially placed at five frnlmg of tissue 4 With further research progress it became evident that the index of 3 or 5 frnlmg was toa low to characterize the degree of hormone dependence of the"neoplastic tissue 5 • This index was gradually elevated to 10, 20 and 30 fm/mg.Even ~ensen6, as his own experience increased, carne to the conclusion that 50 fm/mg would be the ideal index to make a correct ~valuation and prediction of therapeutic response to the endocrine treatment.
Meanwhile, in addition to quantitative methods for receptor dosages, immunohistologic qualitative techniques were developed following the pioneer research started by Pertschuk 7 • When the tumor receptor content was not established during surgery, such information could possibly be determined through paraffin sections.Thus, the hormonal dependence of breast cancer pointed out by the receptor presence, in both estrogen and progesterone conditions, being positive or negative, could represent an altemative approach to improve the mammary carcinoma management.
In this study, we present the results of quantitative biochemical dosages ofhormonal receptors in frozen tumor tissue and quaIitative immunohistochemicaI analyses in paraffin sections carried out on the same group of patients.

MATERIAL ANO METHOOS
Out of a series of 100 women treafed for mammary carcinoma with an asympto'matic survivaI of 5 to 15 years, with receptor dosages for estrogen and progesterone (Dextran-Charcoal method) and with the" immunohistochemical determinations of estrogen and progesterone antibodies (P29 Biogenex), we selected 37 patients presenting reliable paraffin sections.
This group varied in age from 30 to 72 years old; the clinicaI stages were: CS O: The hormonaI receptor values ranged from zero to "762 frnlmg for ER, and from zero to 1,629 fm/mg for PgR.
The positiveness value was set at 30 fm/mg.For qualitative determinations, the resuIts were considered as positive or negative for both conditions.
The median survival rate ofthis series reached 105.5 months.It was analyzed for prognostie purposes and correlated to the clinicaI stage of disease as well with the axillary st~tus and its receptor positiveness, according to both quantitative and qualitative procedures.In clinicaI stage zero with non palpable tumor the survival rate was 120 months.When the axilla was negative 113.7 months.In receptor positive above 30 fmlmg it was 112.7, and only 109.4 months for the patient presenting qualitative positive results.
The comparison between the two methods is shown in Tables 1 and 2. The concordance rate for ER was 64.8% "and for PgR 51 %.Considering that numerous authors accept 20 fm/mg as positive for both hormone receptors, we also used this indexoAs a resuIt, the estrogen receptor showed a reduction of discrepancy from 35.1 to 32%.For the progesterone receptor there was a discrepancy increase to 57%.

OISCUSSION
Jensen 3 noticed that the longer survi vaI and better response rates to hormonal manipuIation were directly proportional to quantitative receptor leveIs.The higher the index the greater the benefit.SimilarIy, adrena,Iectomy and where hormone responsive and nonresponsive cells were present in variable proportions.For Osbome, these varying fractions were responsible for the 15% to 34% non-concordance of his results.He added that the clinicaI responses related to immunohistochemistry were non-satisfactory in 40% of his patients.Borjesson et al 18 carne to the same conclusion.Thorpe et al 19 found that the uniformization of the techniques of citosol preparation contributed to the reduction of the differences seen in the dosages performed by several laboratories, especially concerning the progesterone receptors.
Ozello et al 20 confronted the quantitative method with immunohistochemistry executed in frozen tissues, imprints and paraffin block sections.Except for the latter, the results between the two methods were concordant.
It must be said that one reason for disagreement between quantitative and qualitative methods using frozen sections is the low index criterion for positiveness.ParI and Posey21 verified 89% in concordance when the index for receptors was taken at 3 fentomoles.Kinsel et aI 22using 10 fm/mg as parameter found the agreement just below 70%.
Finally, Hasson et al 23 stressed that the material to be submitted either to quantitative or qualitative methods should be taken early from the biopsy fragment and not from the surgical specimen.Besides, a further measure to reduce the discrepancies is that the tissue should be taken from different areas of the tumor.This precaution applies to either the quantitative or the qualitative determinations.

CONCLUSIONS
Our study of correlation between the quantitative method with d~xtran charcoal technique for ER and PgR and the qualitative immunohistochemical procedure using antibodies antireceptors for estrogen and progesterone on ' paraffin sections, reached the conclusion that the concordance between the two methods is not satisfact~ry.Thus, we do not recommend the immunohistochemical determination applied to paraffin sections as an altemativé to the quantitative procedure for the prognostic evaluation of mammary carcinoma.