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Biphasic Scatchard plots of oestrogen receptors are associated with low pS2 levels in human breast cancers
Cancer Letters 144 (1999) 17±23
Biphasic Scatchard plots of oestrogen receptors are associated with low pS2 levels in human breast cancers S. Marsigliante a,*, L. Biscozzo a, G. Leo b, C. Storelli a a
Dipartimento di Biologia, Laboratorio di Fisiologia, UniversitaÁ di Lecce, Via Provinciale per Monteroni, 73100 Lecce, Italy. b Ospedale `V. Fazzi', Laboratorio di Biologia Molecolare, 73100 Lecce, Italy. Received 2 March 1999; received in revised form 23 April 1999; accepted 23 April 1999
Abstract In a series of 100 breast tumours, oestrogen receptors (ER) were analysed by Scatchard plots, progesterone receptors (PR) by enzyme immunoassay and pS2 by an immunoradiometric assay. Scatchard analysis gave information on receptor heterogeneity in that there was a large variation in Kd values obtained, from 0.001±2.95 nM. This variation was largely con®ned to tumours containing less than 70 fmol receptor per mg protein, while tumours with higher receptor concentrations were a more homogeneous population with low Kd values. An obvious correlation between ER and PR was found; moreover, pS2 was correlated to both ER and PR. In addition, 20 of the 100 tumours gave biphasic Scatchard plots, indicating the presence of at least two oestrogen-binding moieties, with Kd values and concentrations both in the range of those of receptors. The tumours displaying biphasic Scatchard plots had very low pS2 expression, regardless of ER concentrations; this was not true for PR. It is suggested that variability in responses to endocrine therapy may be related to the heterogeneity of the ER present in breast tumours. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Breast cancer; Scatchard analysis; ER; pS2
1. Introduction The requirement to measure oestrogen and progesterone receptors (ER and PR) in breast tumours is generally accepted [1±4]. Several methods have been employed to this end, including ligand-binding assays followed by sucrose density gradient centrifugation, isoelectric focussing separation [5±10], or multi-point assay with data generally analysed by the method of Scatchard [11], which gives information on receptor af®nity as well as concentration; monoclonal antibodies also give valuable results * Corresponding author. Tel.: 139-832-320711; fax: 139-832324-220. E-mail address: [email protected] (S. Marsigliante)
both in an immunoenzymatic form and in an immunocytochemistry assay [12,13]. Clinically, it is generally considered that the key diagnostic information is simply the presence or absence of receptors. However, it is certainly possible that other factors should be taken into account, including receptor af®nities and concentrations. Along with ER, the presence of PR [14,15] and pS2 protein [16±19] are considered to re¯ect a functional mechanism by which the tumour cells are able to respond to oestrogen stimulation. pS2 shows high homology with the two insulin-like growth factors, IGFI and IGFII [20], and, clinically, it can be considered an additional marker of hormone sensitivity. This paper describes studies which show the relationships between receptor number and af®nity and
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00178-0
18
S. Marsigliante et al. / Cancer Letters 144 (1999) 17±23
gives evidence of more than one receptor type in cytosolic preparations of breast tumours. Since the presence of both PR and pS2 would guarantee also the presence of an ER able to activate transcription [21], we describe here further links between ER heterogeneity and pS2 and PR expression. 2. Material and methods 2.1. Tumour specimens All patients were females between 32 and 87 years of age (mean and median age were 56 years). None had received pre-operative tamoxifen therapy. Mammary tumour specimens were obtained at operation and cellular areas of the tissue were placed immediately in liquid nitrogen, transported to the laboratory and stored in liquid nitrogen until processing. 2.2. Tissue preparation High speed tumour supernatants (100 000 £ g) were prepared as follows: tumour tissue was homogenised, using an Ultra-Turrax homogeniser, in glycerol phosphate buffer of low ionic strength (10% glycerol (v/v), 10 mM K2HPO4/KH2PO4, 1.5 mM EDTA, 10 mM MgCl2), containing 1 mg/ml of each of the protease inhibitors soybean trypsin inhibitor, leupeptin and aprotinin, and 1 mM of phenylmethylsulphonyl ¯uoride. The homogenate was centrifuged for 15 min at 900 £ g at 48C. The supernatant was centrifuged again for 60 min at 100 000 £ g and the resulting supernatant (termed `cytosol') was used for pS2, ER and PR determinations. 2.3. Scatchard analysis Aliquots (100 ml) of tumour supernatant were incubated with increasing concentrations of [ 3H]oestradiol (0.04±20 nM) in the presence (competed tubes) or absence (non-competed tubes) of 100-fold excess of diethylstilbestrol (DES) for 18 h at 48C. Free and bound steroids were separated by dextran-coated charcoal (DCC) extraction. A suspension of DCC (500 ml of 0.25% charcoal 0.025% dextran in 10 mM Tris 1.5 mM EDTA buffer (pH 7.4)) was added to each tube, mixed and incubated for 10 min. After centrifugation (5 min, at 10 000 £ g) aliquots (500 ml)
were added to 8 ml liquid scintillation cocktail (Packard). The steroid was extracted into the organic phase by vigorous shaking, and radioactivity measured in a liquid scintillation counter. At all concentrations counts for competed tubes (nonspeci®c binding) were subtracted from counts for non-competed tubes (total binding) to give values for hormones speci®cally bound to receptors. The data are plotted according to the Scatchard method [11], and the appropriate regression line ®tted to the points by the least squares method. Resolution of non-linear Scatchard plots was performed by a dedicated computer using software according to the method of Rosenthal [22] and Zierler [23]. 2.4. Immunoradiometric assay of pS2 The pS2 assay was performed by solid phase two site immunoradiometric assays according to the instructions provided with the CIS Biointernational Kits, Gif-Sur-Yvette, France. This system has been validated by others [24,25] and used for pS2 assay by many research groups [26±28]. 2.5. EIA of progesterone receptors Well-established enzyme immunoassays (EIAs) for PR were used instead of ligand binding procedures, followed by Scatchard analysis, because the volume of cytosol needed for the latter was rarely available due to the small size of the biopsies and the need for other determination procedures. EIA was carried out according to the manufacturer's instructions (Abbot, Chicago, IL). These assays are based on the `sandwich' principle and use two monoclonal antibodies against different epitopes on the PR from MCF-7 breast cancer cells. The ®rst, attached to a glass bead, recognises an epitope between the DNA- and steroid-binding domains. The second antibody, supplied in solution, recognises a sequence close to the steroid-binding domain and is conjugated to horseradish-peroxidase. The receptor present in the specimen binds to the solid phase and also binds the second antibody. The beads are then incubated with an enzyme substrate and the colour developed is a measure of the bound receptor.
S. Marsigliante et al. / Cancer Letters 144 (1999) 17±23
Fig. 1. Biphasic Scatchard plots from a representative ER1ve tumour.
2.6. Protein estimations These were carried out using the method of Bradford [29] using BSA as the standard. 2.7. Statistical analysis Since ER, PR and pS2 data do not conform to Gaussian distributions, we applied non-parametric statistical procedures. Analysis of the correlations between concentration values of steroid receptors and pS2 was carried out using Spearman's rank correlation. The Mann±Whitney U rank sum test was used to test differences in location of the above-mentioned parameters. A probability level of 0.05 or less was chosen to represent statistical signi®cance.
19
greater than 0.9, with the exception of some clearly multicomponent examples (biphasic Scatchard plots found in 20 tumours) described below (Fig. 1). The ER1ve tumours, as shown above, had a wide range of receptor concentrations, and in addition, Scatchard analysis showed a large variation in the dissociation constant (from 0.001±2.95 nM), as shown in Fig. 2. When considering those ER1ve tumours having Scatchard plots with a single receptorial component (n 56), the median ER concentration value was 70 fmol/mg protein. Using this median value as a cut-off, two groups of tumours were obtained, a ®rst with ER # 70 fmol/mg protein and a second with ER . 70 fmol/mg protein. As shown in Fig. 2 tumours with greater than 70 fmol receptor/mg protein had Kd values of 0:094 ^ 0:036 nM (mean ^ SD, n 28), while tumours with less than 70 fmol receptorial protein had Kd values of 0:474 ^ 0:622 nM; these two groups of Kd values were signi®cantly different (P 0:0092 by Mann±Whitney U rank sum test, Fig. 2). Twenty of the 100 tumour supernatants did not give classical Scatchard plots, and some of these are illustrated in Fig. 1. These clearly showed two speci®c oestrogen-binding components which were resolved according to the method of Rosenthal [22] and Zierler [23]. Of these, one had high af®nity, Kd value 0.062 ^ 0.04 nM (n 20), with concentrations ranging from 10±125 fmol/mg protein. The other component was of lower af®nity with a Kd value of 2:59 ^ 0:65 nM (n 20), with concentrations ranging from 19±511 fmol/mg protein.
3. Results Using a cut-off of 10 fmol/mg protein, 76 tumours (76%) were ER positive (ER1ve), with concentrations ranging from 10±602 fmol/mg protein (mean ^ SD: 69^102 fmol/mg protein; median: 34 fmol/ mg protein). Dissociation constants (Kd values) were calculated from the slopes of the regression lines given in the Scatchard plots. In replicated estimations on the tumours the correlation coef®cients (r) were generally
Fig. 2. Box and whiskers representation of Kd values in ER1ve tumours divided into groups of differing ER concentrations (above and below the median). P values obtained by Mann±Whitney Utest. In this representation, the central box covers the middle 50% of the data values, between the upper and lower quartiles. The bars extend out to the extremes, while the central line is at the median. Those values that are beyond 1.5 times the inter-quartile range beyond the central box are plotted as individual points.
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S. Marsigliante et al. / Cancer Letters 144 (1999) 17±23
than those which did not (Mann±Whitney U rank sum test: P 0:004). pS2 was also quantitatively associated with the expression of ER and PR, in that rank correlation by Spearman's gave rS 0:33, (P 0:0012) and rS 0:41, (P 0:0001), respectively (Fig. 3). Overall, pS2 values were not correlated to ER Kd values (rank correlation by Spearman's P 0:086). pS2 values in tumours expressing biphasic Scatchard plots of ER (n 20) were signi®cantly lower (Mann±Whitney U rank sum test: P 0:004) than those found in tumours having one single receptor population (Fig. 4). No differences in PR expression between tumours expressing biphasic Scatchard plots of ER and those that did not were found (Mann±Whitney U rank sum test: P 0:09). 4. Discussion ER has been used as a predictor of prognosis and response to endocrine therapy in breast cancer patients. This is by no means invalidated by the realisation that while originally the interpretation of data assumed that `cytosolic' receptors were estimated, they are now considered to be a pool of receptors readily disassociated from the nucleus by conventional cell fractionation techniques. Moreover, determination of the PR concentration is of equal or greater value than determination of the ER concentra-
Fig. 3. Scatter plots of pS2 and ER (up), pS2 and PR (middle) and ER versus PR (down) in 100 human breast cancer specimens. P values obtained by Spearman's rank correlation.
Regarding the tumours, 72/100 were PR positive (PR1ve), with concentrations ranging from 10±700 fmol/mg protein (mean: 72:6 ^ 123 fmol/mg protein; median: 15 fmol/mg protein) Spearman's rank correlation between ER and PR was rS 0:44 (P , 0:0001). pS2 content ranged from 0±232 ng/mg protein (mean: 23 ^ 2:3 ng/mg protein; median 5.8 ng/mg protein). Tumours expressing ER had higher levels of pS2
Fig. 4. Box and whiskers representation of pS2 concentration values in the groups of ER-negative tumours (ER 0, i.e. ER , 10 fmol/mg protein) and strati®ed in the groups of ER 1 ve (ER $ 10 fmol/mg protein) tumours having different ER status (i.e. those having linear Scatchard plots, and further divided into two groups having ER values above and below the median concentration value, and those expressing biphasic Scatchard plots of ER). P values obtained by Mann±Whitney U-test.
S. Marsigliante et al. / Cancer Letters 144 (1999) 17±23
tion for predicting the disease-free survival of patients and response to endocrine treatment [15]; however, the ER1ve/PR1ve status appears to be the best prognostic factor for assessing response [14,30,31]. Conversely, the pS2 role in predicting prognosis is contradictory [17,18,32,33], but it seems to be associated with a response to hormonal therapy [34]. Clearly, the determination of the pS2 values in patients whose tumours express only one of the two sex steroid receptors could help in the prediction of response to hormonal therapy. The presence of pS2 would guarantee also the presence of an ER able to activate transcription [19], but such information should be also associated with the saturation analysis of ER, if one wishes to ascertain its ligand-dependent nature, and also its heterogeneity. The data presented here suggest that information relating to receptor±steroid af®nities, given by Scatchard analysis, may be valuable. The variation in Kd values (Figs. 1 and 2) shows a remarkable disparity between individual patients, indicating that the practice of recording only the presence or absence of receptors (ER assayed by a single saturating dose or by EIA) may disguise the existence of more than one sub-population. A more homogeneous group is revealed by the separate treatment of tumours containing greater than 70 fmol receptor/mg protein (Fig. 2). This group shows low Kd values within a much smaller range. The relationship between high receptor concentration and high af®nity, for which there is no readily available functional explanation, is interesting; however, it may be due to the nature of the gene and its control in this group. The group of patients whose tumours had receptor concentrations lower than 70 fmol/mg protein showed much more variation in Kd values, suggesting that further subpopulations may exist which cannot be distinguished on the basis of any obvious criterion. The heterogeneity of ER expression is also apparent in individual subjects, in whose tumours biphasic saturation plots and Scatchard plots (Fig. 1) show at least two sites of speci®c hormone binding, either of which alone would in general ®t into the narrow range of concentrations and Kd values characteristic of receptors in tumours. These two sites should not be confused with the types I and II binding sites described by others [35]. In the present case, both binding sites would ®t the category of type I receptor used by
21
these authors, whose type II sites had high concentrations and low Kd values which were outside the range which would be detected in the protocol described here. The values for the different Kd values presented here are more consistent with those given by Weichman [36] calculated for the 4S and 8S con®gurations. A remarkable ®nding seems to be the relationship between biphasic Scatchard plots and low pS2 values (Fig. 4). This relationship between pS2 and heterogeneous ER could be explained by the fact that a direct control of ER on pS2 expression usually exists in breast cancer. This control may be imperfect in cells having a heterogeneous ER, which may lack functionality, a functional ER being able to bind steroids and thereafter initiate transcription of oestrogen-regulated proteins including PR (which remains highly expressed) and pS2 (low expression). pS2 might function as a growth factor, perhaps able to interact by paracrine mechanisms with other cells (also nonresponsive to the oestrogen); from this viewpoint, the involvement of heterogeneous ER1ve tumour cells, lacking the pS2 paracrine mechanisms, may account for the lack of hormonal response. In conclusion, if the limit of diagnostic data required is simply the presence or absence of ER, then either the single saturating dose, the enzyme immunoassay or the Scatchard methods are satisfactory. However, in view of the fact that not all patients with tumours containing ER are responsive to endocrine therapy, further information on the implications of the receptor heterogeneity, as revealed by Scatchard analysis, is also needed.
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S. Marsigliante et al. / Cancer Letters 144 (1999) 17±23 M. Rio, J. Bellocq, P. Chanbon, A.D. Thor, pS2 expression and response to hormonal therapy in patients with advanced breast cancer, Cancer Res. 51 (1991) 624±628. [35] S. Marsigliante, L. Biscozzo, S. Barker, G. Leo, J.R. Puddefoot, G.P. Vinson, C. Storelli, Type II oestrogen binding site is
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Biphasic Scatchard plots of oestrogen receptors are associated with low pS2 levels in human breast cancers S. Marsigliante a,*, L. Biscozzo a, G. Leo b, C. Storelli a a
Dipartimento di Biologia, Laboratorio di Fisiologia, UniversitaÁ di Lecce, Via Provinciale per Monteroni, 73100 Lecce, Italy. b Ospedale `V. Fazzi', Laboratorio di Biologia Molecolare, 73100 Lecce, Italy. Received 2 March 1999; received in revised form 23 April 1999; accepted 23 April 1999
Abstract In a series of 100 breast tumours, oestrogen receptors (ER) were analysed by Scatchard plots, progesterone receptors (PR) by enzyme immunoassay and pS2 by an immunoradiometric assay. Scatchard analysis gave information on receptor heterogeneity in that there was a large variation in Kd values obtained, from 0.001±2.95 nM. This variation was largely con®ned to tumours containing less than 70 fmol receptor per mg protein, while tumours with higher receptor concentrations were a more homogeneous population with low Kd values. An obvious correlation between ER and PR was found; moreover, pS2 was correlated to both ER and PR. In addition, 20 of the 100 tumours gave biphasic Scatchard plots, indicating the presence of at least two oestrogen-binding moieties, with Kd values and concentrations both in the range of those of receptors. The tumours displaying biphasic Scatchard plots had very low pS2 expression, regardless of ER concentrations; this was not true for PR. It is suggested that variability in responses to endocrine therapy may be related to the heterogeneity of the ER present in breast tumours. q 1999 Elsevier Science Ireland Ltd. All rights reserved. Keywords: Breast cancer; Scatchard analysis; ER; pS2
1. Introduction The requirement to measure oestrogen and progesterone receptors (ER and PR) in breast tumours is generally accepted [1±4]. Several methods have been employed to this end, including ligand-binding assays followed by sucrose density gradient centrifugation, isoelectric focussing separation [5±10], or multi-point assay with data generally analysed by the method of Scatchard [11], which gives information on receptor af®nity as well as concentration; monoclonal antibodies also give valuable results * Corresponding author. Tel.: 139-832-320711; fax: 139-832324-220. E-mail address: [email protected] (S. Marsigliante)
both in an immunoenzymatic form and in an immunocytochemistry assay [12,13]. Clinically, it is generally considered that the key diagnostic information is simply the presence or absence of receptors. However, it is certainly possible that other factors should be taken into account, including receptor af®nities and concentrations. Along with ER, the presence of PR [14,15] and pS2 protein [16±19] are considered to re¯ect a functional mechanism by which the tumour cells are able to respond to oestrogen stimulation. pS2 shows high homology with the two insulin-like growth factors, IGFI and IGFII [20], and, clinically, it can be considered an additional marker of hormone sensitivity. This paper describes studies which show the relationships between receptor number and af®nity and
0304-3835/99/$ - see front matter q 1999 Elsevier Science Ireland Ltd. All rights reserved. PII: S 0304-383 5(99)00178-0
18
S. Marsigliante et al. / Cancer Letters 144 (1999) 17±23
gives evidence of more than one receptor type in cytosolic preparations of breast tumours. Since the presence of both PR and pS2 would guarantee also the presence of an ER able to activate transcription [21], we describe here further links between ER heterogeneity and pS2 and PR expression. 2. Material and methods 2.1. Tumour specimens All patients were females between 32 and 87 years of age (mean and median age were 56 years). None had received pre-operative tamoxifen therapy. Mammary tumour specimens were obtained at operation and cellular areas of the tissue were placed immediately in liquid nitrogen, transported to the laboratory and stored in liquid nitrogen until processing. 2.2. Tissue preparation High speed tumour supernatants (100 000 £ g) were prepared as follows: tumour tissue was homogenised, using an Ultra-Turrax homogeniser, in glycerol phosphate buffer of low ionic strength (10% glycerol (v/v), 10 mM K2HPO4/KH2PO4, 1.5 mM EDTA, 10 mM MgCl2), containing 1 mg/ml of each of the protease inhibitors soybean trypsin inhibitor, leupeptin and aprotinin, and 1 mM of phenylmethylsulphonyl ¯uoride. The homogenate was centrifuged for 15 min at 900 £ g at 48C. The supernatant was centrifuged again for 60 min at 100 000 £ g and the resulting supernatant (termed `cytosol') was used for pS2, ER and PR determinations. 2.3. Scatchard analysis Aliquots (100 ml) of tumour supernatant were incubated with increasing concentrations of [ 3H]oestradiol (0.04±20 nM) in the presence (competed tubes) or absence (non-competed tubes) of 100-fold excess of diethylstilbestrol (DES) for 18 h at 48C. Free and bound steroids were separated by dextran-coated charcoal (DCC) extraction. A suspension of DCC (500 ml of 0.25% charcoal 0.025% dextran in 10 mM Tris 1.5 mM EDTA buffer (pH 7.4)) was added to each tube, mixed and incubated for 10 min. After centrifugation (5 min, at 10 000 £ g) aliquots (500 ml)
were added to 8 ml liquid scintillation cocktail (Packard). The steroid was extracted into the organic phase by vigorous shaking, and radioactivity measured in a liquid scintillation counter. At all concentrations counts for competed tubes (nonspeci®c binding) were subtracted from counts for non-competed tubes (total binding) to give values for hormones speci®cally bound to receptors. The data are plotted according to the Scatchard method [11], and the appropriate regression line ®tted to the points by the least squares method. Resolution of non-linear Scatchard plots was performed by a dedicated computer using software according to the method of Rosenthal [22] and Zierler [23]. 2.4. Immunoradiometric assay of pS2 The pS2 assay was performed by solid phase two site immunoradiometric assays according to the instructions provided with the CIS Biointernational Kits, Gif-Sur-Yvette, France. This system has been validated by others [24,25] and used for pS2 assay by many research groups [26±28]. 2.5. EIA of progesterone receptors Well-established enzyme immunoassays (EIAs) for PR were used instead of ligand binding procedures, followed by Scatchard analysis, because the volume of cytosol needed for the latter was rarely available due to the small size of the biopsies and the need for other determination procedures. EIA was carried out according to the manufacturer's instructions (Abbot, Chicago, IL). These assays are based on the `sandwich' principle and use two monoclonal antibodies against different epitopes on the PR from MCF-7 breast cancer cells. The ®rst, attached to a glass bead, recognises an epitope between the DNA- and steroid-binding domains. The second antibody, supplied in solution, recognises a sequence close to the steroid-binding domain and is conjugated to horseradish-peroxidase. The receptor present in the specimen binds to the solid phase and also binds the second antibody. The beads are then incubated with an enzyme substrate and the colour developed is a measure of the bound receptor.
S. Marsigliante et al. / Cancer Letters 144 (1999) 17±23
Fig. 1. Biphasic Scatchard plots from a representative ER1ve tumour.
2.6. Protein estimations These were carried out using the method of Bradford [29] using BSA as the standard. 2.7. Statistical analysis Since ER, PR and pS2 data do not conform to Gaussian distributions, we applied non-parametric statistical procedures. Analysis of the correlations between concentration values of steroid receptors and pS2 was carried out using Spearman's rank correlation. The Mann±Whitney U rank sum test was used to test differences in location of the above-mentioned parameters. A probability level of 0.05 or less was chosen to represent statistical signi®cance.
19
greater than 0.9, with the exception of some clearly multicomponent examples (biphasic Scatchard plots found in 20 tumours) described below (Fig. 1). The ER1ve tumours, as shown above, had a wide range of receptor concentrations, and in addition, Scatchard analysis showed a large variation in the dissociation constant (from 0.001±2.95 nM), as shown in Fig. 2. When considering those ER1ve tumours having Scatchard plots with a single receptorial component (n 56), the median ER concentration value was 70 fmol/mg protein. Using this median value as a cut-off, two groups of tumours were obtained, a ®rst with ER # 70 fmol/mg protein and a second with ER . 70 fmol/mg protein. As shown in Fig. 2 tumours with greater than 70 fmol receptor/mg protein had Kd values of 0:094 ^ 0:036 nM (mean ^ SD, n 28), while tumours with less than 70 fmol receptorial protein had Kd values of 0:474 ^ 0:622 nM; these two groups of Kd values were signi®cantly different (P 0:0092 by Mann±Whitney U rank sum test, Fig. 2). Twenty of the 100 tumour supernatants did not give classical Scatchard plots, and some of these are illustrated in Fig. 1. These clearly showed two speci®c oestrogen-binding components which were resolved according to the method of Rosenthal [22] and Zierler [23]. Of these, one had high af®nity, Kd value 0.062 ^ 0.04 nM (n 20), with concentrations ranging from 10±125 fmol/mg protein. The other component was of lower af®nity with a Kd value of 2:59 ^ 0:65 nM (n 20), with concentrations ranging from 19±511 fmol/mg protein.
3. Results Using a cut-off of 10 fmol/mg protein, 76 tumours (76%) were ER positive (ER1ve), with concentrations ranging from 10±602 fmol/mg protein (mean ^ SD: 69^102 fmol/mg protein; median: 34 fmol/ mg protein). Dissociation constants (Kd values) were calculated from the slopes of the regression lines given in the Scatchard plots. In replicated estimations on the tumours the correlation coef®cients (r) were generally
Fig. 2. Box and whiskers representation of Kd values in ER1ve tumours divided into groups of differing ER concentrations (above and below the median). P values obtained by Mann±Whitney Utest. In this representation, the central box covers the middle 50% of the data values, between the upper and lower quartiles. The bars extend out to the extremes, while the central line is at the median. Those values that are beyond 1.5 times the inter-quartile range beyond the central box are plotted as individual points.
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than those which did not (Mann±Whitney U rank sum test: P 0:004). pS2 was also quantitatively associated with the expression of ER and PR, in that rank correlation by Spearman's gave rS 0:33, (P 0:0012) and rS 0:41, (P 0:0001), respectively (Fig. 3). Overall, pS2 values were not correlated to ER Kd values (rank correlation by Spearman's P 0:086). pS2 values in tumours expressing biphasic Scatchard plots of ER (n 20) were signi®cantly lower (Mann±Whitney U rank sum test: P 0:004) than those found in tumours having one single receptor population (Fig. 4). No differences in PR expression between tumours expressing biphasic Scatchard plots of ER and those that did not were found (Mann±Whitney U rank sum test: P 0:09). 4. Discussion ER has been used as a predictor of prognosis and response to endocrine therapy in breast cancer patients. This is by no means invalidated by the realisation that while originally the interpretation of data assumed that `cytosolic' receptors were estimated, they are now considered to be a pool of receptors readily disassociated from the nucleus by conventional cell fractionation techniques. Moreover, determination of the PR concentration is of equal or greater value than determination of the ER concentra-
Fig. 3. Scatter plots of pS2 and ER (up), pS2 and PR (middle) and ER versus PR (down) in 100 human breast cancer specimens. P values obtained by Spearman's rank correlation.
Regarding the tumours, 72/100 were PR positive (PR1ve), with concentrations ranging from 10±700 fmol/mg protein (mean: 72:6 ^ 123 fmol/mg protein; median: 15 fmol/mg protein) Spearman's rank correlation between ER and PR was rS 0:44 (P , 0:0001). pS2 content ranged from 0±232 ng/mg protein (mean: 23 ^ 2:3 ng/mg protein; median 5.8 ng/mg protein). Tumours expressing ER had higher levels of pS2
Fig. 4. Box and whiskers representation of pS2 concentration values in the groups of ER-negative tumours (ER 0, i.e. ER , 10 fmol/mg protein) and strati®ed in the groups of ER 1 ve (ER $ 10 fmol/mg protein) tumours having different ER status (i.e. those having linear Scatchard plots, and further divided into two groups having ER values above and below the median concentration value, and those expressing biphasic Scatchard plots of ER). P values obtained by Mann±Whitney U-test.
S. Marsigliante et al. / Cancer Letters 144 (1999) 17±23
tion for predicting the disease-free survival of patients and response to endocrine treatment [15]; however, the ER1ve/PR1ve status appears to be the best prognostic factor for assessing response [14,30,31]. Conversely, the pS2 role in predicting prognosis is contradictory [17,18,32,33], but it seems to be associated with a response to hormonal therapy [34]. Clearly, the determination of the pS2 values in patients whose tumours express only one of the two sex steroid receptors could help in the prediction of response to hormonal therapy. The presence of pS2 would guarantee also the presence of an ER able to activate transcription [19], but such information should be also associated with the saturation analysis of ER, if one wishes to ascertain its ligand-dependent nature, and also its heterogeneity. The data presented here suggest that information relating to receptor±steroid af®nities, given by Scatchard analysis, may be valuable. The variation in Kd values (Figs. 1 and 2) shows a remarkable disparity between individual patients, indicating that the practice of recording only the presence or absence of receptors (ER assayed by a single saturating dose or by EIA) may disguise the existence of more than one sub-population. A more homogeneous group is revealed by the separate treatment of tumours containing greater than 70 fmol receptor/mg protein (Fig. 2). This group shows low Kd values within a much smaller range. The relationship between high receptor concentration and high af®nity, for which there is no readily available functional explanation, is interesting; however, it may be due to the nature of the gene and its control in this group. The group of patients whose tumours had receptor concentrations lower than 70 fmol/mg protein showed much more variation in Kd values, suggesting that further subpopulations may exist which cannot be distinguished on the basis of any obvious criterion. The heterogeneity of ER expression is also apparent in individual subjects, in whose tumours biphasic saturation plots and Scatchard plots (Fig. 1) show at least two sites of speci®c hormone binding, either of which alone would in general ®t into the narrow range of concentrations and Kd values characteristic of receptors in tumours. These two sites should not be confused with the types I and II binding sites described by others [35]. In the present case, both binding sites would ®t the category of type I receptor used by
21
these authors, whose type II sites had high concentrations and low Kd values which were outside the range which would be detected in the protocol described here. The values for the different Kd values presented here are more consistent with those given by Weichman [36] calculated for the 4S and 8S con®gurations. A remarkable ®nding seems to be the relationship between biphasic Scatchard plots and low pS2 values (Fig. 4). This relationship between pS2 and heterogeneous ER could be explained by the fact that a direct control of ER on pS2 expression usually exists in breast cancer. This control may be imperfect in cells having a heterogeneous ER, which may lack functionality, a functional ER being able to bind steroids and thereafter initiate transcription of oestrogen-regulated proteins including PR (which remains highly expressed) and pS2 (low expression). pS2 might function as a growth factor, perhaps able to interact by paracrine mechanisms with other cells (also nonresponsive to the oestrogen); from this viewpoint, the involvement of heterogeneous ER1ve tumour cells, lacking the pS2 paracrine mechanisms, may account for the lack of hormonal response. In conclusion, if the limit of diagnostic data required is simply the presence or absence of ER, then either the single saturating dose, the enzyme immunoassay or the Scatchard methods are satisfactory. However, in view of the fact that not all patients with tumours containing ER are responsive to endocrine therapy, further information on the implications of the receptor heterogeneity, as revealed by Scatchard analysis, is also needed.
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