Annah of Oncology 3: 71-77, 1992. © 1992 Kluwer Academic Publishers. Printed in the Netherlands.
Original article Immunocytochemical determination of the estrogen-regulated proteins M r 24,000, M r 52,000 and DF3 breast cancer associated antigen: Clinical value in advanced breast cancer and correlation with estrogen receptor L. Damstrup,1 J. Andersen,2 D. W. Kufe,3 D. F. Hayes3 & H. Skovgaard Poulsen 24 1 Pathological Anatomical Institute, University of Copenhagen;2 The Receptor Laboratory, Danish Cancer Society, Department of Experimental Clinical Oncology and Department of Oncology, Radiumstationen, Aarhus, Denmark;3Dana-Farber Cancer Institute, Boston, USA;4 Department of Oncology, Rigshospitalet, Copenhagen, Denmark
Summary. The M, 24,000 and Mr 52,000 estrogen-regulated cytosol proteins, and the breast cancer-associated antigen DF3 have been studied in an immunocytochemical assay. Primary tumor specimens from 119 patients with advanced breast cancer who received endocrine therapy have been studied. Monoclonal antibodies were used for the detection of the proteins in formalin-fixed paraffin-embedded blocks. No correlation between Mr 52,000-positive specimens and the presence of estrogen receptor (ER) could be established (p = 0.87, chi-square test) whereas a statistically significant association between Mr 24,000 (p - 0.0002), DF3 antigen (p = 0.044) and ER was demonstrated. No intercorrelation was found between Mr 24,000 and Mr 52,000 or DF3 (p = 0.63, 0.98 and 0.12 respectively). Clinical response was evaluated for immunocytochemical findings, Mr 24,000 (p - 0.37), Mr 52,000 (p - 0.61) and DF3 (p - 0.68) showed
Introduction
Breast cancer is a large, heterogeneous group of malignant diseases. Analysis for estrogen receptors at the initial surgical procedure has been widely accepted practice over the past decade. Clinical response to hormonal treatment either as first-line therapy or at recurrence is very often predicted by the levels of estrogen (ER) and progesterone (PgR) receptors in the malignant tissue [1, 2]. However, it is obvious that other parameters play a substantial role in the growth of breast cancer cells, as a considerable number of receptor-positive tumors do not respond to endocrine therapy [3]. On the basis of this disturbing therapeutical enigma, several markers have been evaluated in order to provide clinicians with additional parameters for the outcome of endocrine therapy in ER-positive patients. Edward et al. [4, 5] have identified and characterized a cytosolic estrogen-regulated M, 24,000 (p24) protein in MCF-7 cells, used to raise a monoclonal antibody (MoAb) against p24. A significant correlation between this protein and the presence of estrogen receptors in malignant breast tumors has been established [6]. The p24 is not commonly expressed in normal breast tissue,
no association whereas ER was statistically correlated (p — 0.00005). Neither overall survival nor disease-free survival correlated to Mr 24,000 (p - 0.18 and 0.75 respectively, logrank test), Mr 52,000 (p = 0.095 and 0.38), or DF3 (p = 0.22 and 0.13) staining, whereas ER-positive tumors did (p — 0.00005). Discrimination between ER-positive responders and ER-positive non-responders was not possible using either Mr 52,000, Mr 24,000 or DF3 staining. Based on our findings we conclude that immunocytochemical staining for Mr 52,000, Mr 24,000 or DF3 cannot be used as a marker to predict response to endocrine therapy in patients with advanced or recurrent breast cancer.
Key words: breast cancer, DF3 antigen, Mr 24,000 protein, Mr 52,000 protein, response to endocrine therapy
lactating breast or in receptor-negative tumors [7, 8]. Garcia et al. [9] have identified and characterized a Mr 52,000 protein (K52), the secretion of which appears to be estrogen-stimulated. MoAb directed against 52K have been raised from a MCF-7 cell lysate [10]. Finally a MoAb (DF3) against a high-molecular-weight mucin-like protein Mr 330,000450,000 has been raised from a membrane-enriched cell extract from a human breast carcinoma liver metastasis [11]. The protein designated CA 15-3 has also been detected in human milk [12]. Cytosolic staining is observed in 70%-80% of breast carcinomas, whereas staining is seen in only approximately 10% of benign breast tumors [13]. Furthermore, serial CA 15-3 screening of serum, employing DF3, in patients with breast cancer has revealed that recurrence is preceded by increasing CA 15-3 levels [14]. The aim of this study was to evaluate whether these monoclonal antibodies could be used as markers for the prediction of response to endocrine therapy of advanced breast cancer, and to evaluate whether any of these monoclonal antibodies could discriminate between ER-positive responders and ER-positive nonresponders.
72 Table 1. Patient characteristics.
(OS) and disease-free survival (DFS) were recorded from the date of operation until death or recurrence.
Characteristics
ER
p24
K52
DF3
Total no biopsies Analysis failed Age (yrs), median (range)
113 8 68 (29-96) 8 21 29 47
113 10 67 (29-96) 8 22 29 44
113 24 68 (29-96) 8 20 24 37
119 0 68 (29-96) 12 22 35 50
Statistical methods
70 Menopausal status" Premenopausal Postmenopausal Primary surgical treatment Mastectomy (simple) (a.m Cade) Biopsy only Tumorectomy Tumor size 5cm Unknown Histology Infiltrating ductal carcinoma Lobular carcinoma Other subtypes Primary extent of disease Locoregional Metastatic Node negative Node positive Unknown Primary locoregional treatment Radiotherapy None Site of recurrence Soft tissue only Bones and/or soft tissue Viscera and/or soft tissue and/or bones Treatment Tamoxifen Oophorectomy Oophorectomy and tamoxifen Fluoxymestrone Other endocrine treatment
10 95
10 93
10 37
15 104
39 60 5
40 58 4
37 47 4 1
38 73 7 1
1
1
54 29
54 28
45 24
64 31 24
90 7 8
88 7 8
75 5 9
105 7 7
88 17 23 37 45
86 17 21 37 45
76 13 18 29 42
102 17 27 46 46
66 49
65 48
56 33
73 46
40 27 38
40 25 38
35 21 33
40 28 51
82 5 5 2 II
79 5 5 2 12
69 5 5 2 8
92 7 6 2 12
• Menopausal defined as cessation of normal menstruation >5 years before recurrence.
Patients and methods
Differences in observed response to endocrine therapy and status of the different examined parameters was evaluated using x2 test with Yates correction. Differences in DFS and OS were evaluated by the logrank test (16] and Gehan's generalized Wilcoxon test [17]. No differences in DFS or OS were found in any of the cases when analysed by these two statistical methods. All p-values indicated in the text and figures are from the logrank test. Antibodies MoAb antibodies against p24 were kindly provided by Dr. W. L. McGuire; their production and characterization has been described (18, 19]. MoAb antibodies against 52K were purchased from BioSys, France. The production and characterization of 52K has previously been described [9J. DF3 MoAb generation and purification by protein A affinity chromatography has been described [11|. H222 MoAb against ER was purchased from Abbot Laboratories, USA. Immunocytochemtstry Archive formalin-fixed paraffin-embedded biopsies from primary breast tumors were analyzed. Analysis for p24, 52K and ER was done on 113 biopsies. The immunocytochemical technique used for detection of p24, K52 and ER was described previously [20-23]. The DF3 antigen was detected by an immunoperoxidase method on 119 biopsies, and a new ER determination was performed on the same specimens. The results from the new determination were used only to evaluate the correlation between ER and DF3. After deparaffinization and rehydration, slides were washed in water for 5 min and incubated for 10 min with H^Oj/ethanol to block endogenous peroxidase. The reaction was stopped by washing in TRIS/PBS for 5 min. Slides were then incubated with normal goat serum for 20 min. Excess incubation media was poured off and slides covered with MoAb 1 ng/ml for 30 min. The slides were then washed twice with Tris/PBS and incubated with biotinylated goat-anti mouse 35 u.g/ml for 30 min. After two Tris/PBS washes, slides were incubated with Avidin-Biotin-Peroxidase-Complex for 30 min and washed with Tris/PBS for 2 x 1 0 min. Slides were then stained with carbazole for 15 min and rinsed in water. Finally slides were slightly counterstained with haematoxylin for 5 min, washed for 10 min and mounted with glycerol/gelatine. For every biopsy a parallel control was run in which the primary antibody was replaced with normal mouse IgG; furthermore, known positive and negative specimens were also run. Positive staining was defined as cytoplasmic stain of malignant cells in the presence of the primary antibody and negative staining in the absence of the antibody.
Patients One hundred-nineteen patients with primary breast carcinomas, admitted to the department of Oncology, Aarhus Hospital, who had the following characteristics, were included in this study: 1) recurrent or advanced breast cancer with evaluable parameters; 2) no prior systemic antineoplastic treatment; 3) evaluable disease treated with hormones only. At the time of recurrence 104 (87%) of them were postmenopausal. The major sites of recurrence were in decreasing order of priority: viscera, bone and soft tissue. Local recurrence was confirmed histologically and back-dated to the time of discovery. Relapses in bone were dated from the first abnormal X-ray. Recurrence in pleural effusions was accepted with malignant cytology. Characteristic changes in isotope or ultrasound liver scan were also accepted. Endocrine therapy was given predominantly as tamoxifen in 79%. Further patients details are given in Table 1. Criterion of response followed UICC recommendations [15]. Overall survival
Table 2. Correlation of p24, 52K and DF3 with ER status. ER status Positive
Negative
p24 Positive p24 Negative n-99
50 17
11 21
52K Positive 52K Negative n-89
21 42
DF3 Positive DF3 Negative n-114
34 46
p - 0.0002 8 18 p - 0.98 7 27 p - 0.044
73 Table 3. Correlation of p24, 52K and DF3 with response to endocrine therapy.
OS
Response NC + PD
p24 Positive p24 Negative n-103
29 14
34 26
52K Positive 52K Negative n-89
14 15
DF3 Positive DF3 Negative n - 119
14 31
ER Positive ER Negative n=105
40 3
P -0.37
24 36 P -0.61
O
1 % LJ
30 44 P -0.40
M
T
32 30 D —
1 proportion st
CR + PR
Z •x.
0.00005
16
Table 4. Correlation of p24, 52K and DF3 with response in patients with ER-positive tumors.
DFS
Response
1.0 P = 0.54
0.8
p24 Positive p24 Negative n = 68
27 11
23 7
0.6
p - 0.81
52K Positive 52K Negative n-63
13 22
DF3 Positive DF3 Negative n-80
12 27
P24+
8 20 p - 0.65 22 19
p - 0.07
Q.
0.4 • P24-
•s
1
0.2
•
0.0 100
-
50
Results Positive staining for either p24, 52K or DF3 antigen was confined to the cytoplasm. AJ1 positive slides contained a mixture of stained and unstained cells. This variation was seen in both adjacent cells and as an area to area difference. The degree of staining was quantified for p24 and 52K as none, + (50% positive). In this study 2+ or 3+ was regarded as positive and 0 or 1+ as negative. The staining for DF3 antigen was quantified as 0 (none), 1 (pale), 2 (good), 3 (strong), or 4 (intense). For the purpose of this study 3 and 4 were quantified as positive, and 0-2 as negative. Slides with any degree of specific staining with the ER antibody were classified as positive. Mr 24,000protein studies
One hundred-thirteen slides were studied with p24 antibodies of which 10 were technically unevaluable. Of the remaining 103 cases, 63 (61%) were positive and 40 (39%) negative. A statistical correlation between p24 staining and ER was found in the 99 patients for
0
^
10
12
Study year
Fig. 1. Overall Survival (OS) and Disease-Free Survival (DFS) in 103 patients with advanced breast cancer with respect to p24 staining. P-values determined by logrank test.
whom receptor data were available (p - 0.0002) (Table 2). Response (complete and partial remission) to endocrine therapy according to UICC criteria [15] was registered in 43 patients (42%) and no response (no change and progressive disease) in 60 patients (58%). No relationship between response and p24 staining was found (p = 0.37) (Table 3). Despite the significant relationship between p24 and ER no association between p24 and response in the 68 ER-positive patients was found (p = 0.81) (Table 4). Overall survival (p = 0.076) and disease-free survival (p — 0.54) were not related to p24 (Fig. 1). Mr 52,000protein studies
In the 89 slides successfully analyzed, 29 (33%) were positive and 60 (67%) negative. No correlation be-
74 OS
OS 1.0 P = 0.22
0.8 DF3 3-4
1E
\
0.6
DF3 0 - 2 "
0.4 0.2 0.0 100 50
~
0 6
14
8
-^_
^
12
14
10
16
Study year
DFS
DFS 1.0
P = 0.13
0.8
s
1c
0.6
o
a. 8
DF3 3-4 0.4
a.
j
DF3 0-2
^
0.2
•
= ^
1^ -1
0.0
,
i
'
1
100 50 0 6
14
8
10
12
14
Study y«ar
Fig. 2. Overall Survival (OS) and Disease-Free Survival (DFS) in 89 patients with advanced breast cancer with respect to 52K staining. P-values determined by logrank test.
Fig. 3. Overall Survival (OS) and Disease-Free Survival (DFS) in 119 patients with advanced breast cancer with respect to DF3 staining. P-values determined by logrank test.
tween 52K or ER status could be established (p = 0.98) with receptor data available in all patients (Table 2). Response was registered in 38 patients (43%) and nonresponse in 51 patients (57%). The relationship between 52K and response (p = 0.61) is shown in Table 3. The association between response to endocrine therapy and 52K status in 63 patients with ER-positive tumors is given in Table 4 (p = 0.65). Overall survival (p - 0.018) and disease-free survival (p - 0.22) in 52Kpositive and -negative patients is seen in Fig. 2.
A significant correlation (p = 0.044) was established between DF3-positive tumors and ER-positive tumors (Table 2). Response was registered in 45 patients (38%) and non-response (NC+PD) in 74 patients (62%) (p — 0.40) (Table 3). No relationship was found between response to endocrine therapy and DF3 status in 80 patients with ER-positive tumors (p = 0.07) (Table 4). Overall survival (p = 0.22) and disease-free survival (p - 0.13) in DF3-positive and -negative patients is seen in Fig. 3. No inter-relationship between 52K, p24 and DF3 status was found in our material (Table 5).
DF3 studies Estrogen receptor studies
One hundred-nineteen slides were examined for DF3 staining: 44 (37%) of them were positive and 75 (63%) were negative. The same slides were also examined for the presence of ER, which was found in 114 cases.
One hundred-five slides were examined for the presence of ER: 72 (69%) were found to be positive and 33 (31%) negative. Forty 78 14 7 Negative 15 8 7 11 17 10 pmol/mg cytosol protein) and ER status in 242 pre/ 081 p value 0.63 0.S9 0.94 Tumor size perimenopausal women. However, they found no cor>5 cm 17 12 17 11 6 18 22 12 relation between 52K and ER status in postmeno17 28 years 36 22 35 23 14 38 study found overall 52K status uncorrelated to ER
Original article Immunocytochemical determination of the estrogen-regulated proteins M r 24,000, M r 52,000 and DF3 breast cancer associated antigen: Clinical value in advanced breast cancer and correlation with estrogen receptor L. Damstrup,1 J. Andersen,2 D. W. Kufe,3 D. F. Hayes3 & H. Skovgaard Poulsen 24 1 Pathological Anatomical Institute, University of Copenhagen;2 The Receptor Laboratory, Danish Cancer Society, Department of Experimental Clinical Oncology and Department of Oncology, Radiumstationen, Aarhus, Denmark;3Dana-Farber Cancer Institute, Boston, USA;4 Department of Oncology, Rigshospitalet, Copenhagen, Denmark
Summary. The M, 24,000 and Mr 52,000 estrogen-regulated cytosol proteins, and the breast cancer-associated antigen DF3 have been studied in an immunocytochemical assay. Primary tumor specimens from 119 patients with advanced breast cancer who received endocrine therapy have been studied. Monoclonal antibodies were used for the detection of the proteins in formalin-fixed paraffin-embedded blocks. No correlation between Mr 52,000-positive specimens and the presence of estrogen receptor (ER) could be established (p = 0.87, chi-square test) whereas a statistically significant association between Mr 24,000 (p - 0.0002), DF3 antigen (p = 0.044) and ER was demonstrated. No intercorrelation was found between Mr 24,000 and Mr 52,000 or DF3 (p = 0.63, 0.98 and 0.12 respectively). Clinical response was evaluated for immunocytochemical findings, Mr 24,000 (p - 0.37), Mr 52,000 (p - 0.61) and DF3 (p - 0.68) showed
Introduction
Breast cancer is a large, heterogeneous group of malignant diseases. Analysis for estrogen receptors at the initial surgical procedure has been widely accepted practice over the past decade. Clinical response to hormonal treatment either as first-line therapy or at recurrence is very often predicted by the levels of estrogen (ER) and progesterone (PgR) receptors in the malignant tissue [1, 2]. However, it is obvious that other parameters play a substantial role in the growth of breast cancer cells, as a considerable number of receptor-positive tumors do not respond to endocrine therapy [3]. On the basis of this disturbing therapeutical enigma, several markers have been evaluated in order to provide clinicians with additional parameters for the outcome of endocrine therapy in ER-positive patients. Edward et al. [4, 5] have identified and characterized a cytosolic estrogen-regulated M, 24,000 (p24) protein in MCF-7 cells, used to raise a monoclonal antibody (MoAb) against p24. A significant correlation between this protein and the presence of estrogen receptors in malignant breast tumors has been established [6]. The p24 is not commonly expressed in normal breast tissue,
no association whereas ER was statistically correlated (p — 0.00005). Neither overall survival nor disease-free survival correlated to Mr 24,000 (p - 0.18 and 0.75 respectively, logrank test), Mr 52,000 (p = 0.095 and 0.38), or DF3 (p = 0.22 and 0.13) staining, whereas ER-positive tumors did (p — 0.00005). Discrimination between ER-positive responders and ER-positive non-responders was not possible using either Mr 52,000, Mr 24,000 or DF3 staining. Based on our findings we conclude that immunocytochemical staining for Mr 52,000, Mr 24,000 or DF3 cannot be used as a marker to predict response to endocrine therapy in patients with advanced or recurrent breast cancer.
Key words: breast cancer, DF3 antigen, Mr 24,000 protein, Mr 52,000 protein, response to endocrine therapy
lactating breast or in receptor-negative tumors [7, 8]. Garcia et al. [9] have identified and characterized a Mr 52,000 protein (K52), the secretion of which appears to be estrogen-stimulated. MoAb directed against 52K have been raised from a MCF-7 cell lysate [10]. Finally a MoAb (DF3) against a high-molecular-weight mucin-like protein Mr 330,000450,000 has been raised from a membrane-enriched cell extract from a human breast carcinoma liver metastasis [11]. The protein designated CA 15-3 has also been detected in human milk [12]. Cytosolic staining is observed in 70%-80% of breast carcinomas, whereas staining is seen in only approximately 10% of benign breast tumors [13]. Furthermore, serial CA 15-3 screening of serum, employing DF3, in patients with breast cancer has revealed that recurrence is preceded by increasing CA 15-3 levels [14]. The aim of this study was to evaluate whether these monoclonal antibodies could be used as markers for the prediction of response to endocrine therapy of advanced breast cancer, and to evaluate whether any of these monoclonal antibodies could discriminate between ER-positive responders and ER-positive nonresponders.
72 Table 1. Patient characteristics.
(OS) and disease-free survival (DFS) were recorded from the date of operation until death or recurrence.
Characteristics
ER
p24
K52
DF3
Total no biopsies Analysis failed Age (yrs), median (range)
113 8 68 (29-96) 8 21 29 47
113 10 67 (29-96) 8 22 29 44
113 24 68 (29-96) 8 20 24 37
119 0 68 (29-96) 12 22 35 50
Statistical methods
70 Menopausal status" Premenopausal Postmenopausal Primary surgical treatment Mastectomy (simple) (a.m Cade) Biopsy only Tumorectomy Tumor size 5cm Unknown Histology Infiltrating ductal carcinoma Lobular carcinoma Other subtypes Primary extent of disease Locoregional Metastatic Node negative Node positive Unknown Primary locoregional treatment Radiotherapy None Site of recurrence Soft tissue only Bones and/or soft tissue Viscera and/or soft tissue and/or bones Treatment Tamoxifen Oophorectomy Oophorectomy and tamoxifen Fluoxymestrone Other endocrine treatment
10 95
10 93
10 37
15 104
39 60 5
40 58 4
37 47 4 1
38 73 7 1
1
1
54 29
54 28
45 24
64 31 24
90 7 8
88 7 8
75 5 9
105 7 7
88 17 23 37 45
86 17 21 37 45
76 13 18 29 42
102 17 27 46 46
66 49
65 48
56 33
73 46
40 27 38
40 25 38
35 21 33
40 28 51
82 5 5 2 II
79 5 5 2 12
69 5 5 2 8
92 7 6 2 12
• Menopausal defined as cessation of normal menstruation >5 years before recurrence.
Patients and methods
Differences in observed response to endocrine therapy and status of the different examined parameters was evaluated using x2 test with Yates correction. Differences in DFS and OS were evaluated by the logrank test (16] and Gehan's generalized Wilcoxon test [17]. No differences in DFS or OS were found in any of the cases when analysed by these two statistical methods. All p-values indicated in the text and figures are from the logrank test. Antibodies MoAb antibodies against p24 were kindly provided by Dr. W. L. McGuire; their production and characterization has been described (18, 19]. MoAb antibodies against 52K were purchased from BioSys, France. The production and characterization of 52K has previously been described [9J. DF3 MoAb generation and purification by protein A affinity chromatography has been described [11|. H222 MoAb against ER was purchased from Abbot Laboratories, USA. Immunocytochemtstry Archive formalin-fixed paraffin-embedded biopsies from primary breast tumors were analyzed. Analysis for p24, 52K and ER was done on 113 biopsies. The immunocytochemical technique used for detection of p24, K52 and ER was described previously [20-23]. The DF3 antigen was detected by an immunoperoxidase method on 119 biopsies, and a new ER determination was performed on the same specimens. The results from the new determination were used only to evaluate the correlation between ER and DF3. After deparaffinization and rehydration, slides were washed in water for 5 min and incubated for 10 min with H^Oj/ethanol to block endogenous peroxidase. The reaction was stopped by washing in TRIS/PBS for 5 min. Slides were then incubated with normal goat serum for 20 min. Excess incubation media was poured off and slides covered with MoAb 1 ng/ml for 30 min. The slides were then washed twice with Tris/PBS and incubated with biotinylated goat-anti mouse 35 u.g/ml for 30 min. After two Tris/PBS washes, slides were incubated with Avidin-Biotin-Peroxidase-Complex for 30 min and washed with Tris/PBS for 2 x 1 0 min. Slides were then stained with carbazole for 15 min and rinsed in water. Finally slides were slightly counterstained with haematoxylin for 5 min, washed for 10 min and mounted with glycerol/gelatine. For every biopsy a parallel control was run in which the primary antibody was replaced with normal mouse IgG; furthermore, known positive and negative specimens were also run. Positive staining was defined as cytoplasmic stain of malignant cells in the presence of the primary antibody and negative staining in the absence of the antibody.
Patients One hundred-nineteen patients with primary breast carcinomas, admitted to the department of Oncology, Aarhus Hospital, who had the following characteristics, were included in this study: 1) recurrent or advanced breast cancer with evaluable parameters; 2) no prior systemic antineoplastic treatment; 3) evaluable disease treated with hormones only. At the time of recurrence 104 (87%) of them were postmenopausal. The major sites of recurrence were in decreasing order of priority: viscera, bone and soft tissue. Local recurrence was confirmed histologically and back-dated to the time of discovery. Relapses in bone were dated from the first abnormal X-ray. Recurrence in pleural effusions was accepted with malignant cytology. Characteristic changes in isotope or ultrasound liver scan were also accepted. Endocrine therapy was given predominantly as tamoxifen in 79%. Further patients details are given in Table 1. Criterion of response followed UICC recommendations [15]. Overall survival
Table 2. Correlation of p24, 52K and DF3 with ER status. ER status Positive
Negative
p24 Positive p24 Negative n-99
50 17
11 21
52K Positive 52K Negative n-89
21 42
DF3 Positive DF3 Negative n-114
34 46
p - 0.0002 8 18 p - 0.98 7 27 p - 0.044
73 Table 3. Correlation of p24, 52K and DF3 with response to endocrine therapy.
OS
Response NC + PD
p24 Positive p24 Negative n-103
29 14
34 26
52K Positive 52K Negative n-89
14 15
DF3 Positive DF3 Negative n - 119
14 31
ER Positive ER Negative n=105
40 3
P -0.37
24 36 P -0.61
O
1 % LJ
30 44 P -0.40
M
T
32 30 D —
1 proportion st
CR + PR
Z •x.
0.00005
16
Table 4. Correlation of p24, 52K and DF3 with response in patients with ER-positive tumors.
DFS
Response
1.0 P = 0.54
0.8
p24 Positive p24 Negative n = 68
27 11
23 7
0.6
p - 0.81
52K Positive 52K Negative n-63
13 22
DF3 Positive DF3 Negative n-80
12 27
P24+
8 20 p - 0.65 22 19
p - 0.07
Q.
0.4 • P24-
•s
1
0.2
•
0.0 100
-
50
Results Positive staining for either p24, 52K or DF3 antigen was confined to the cytoplasm. AJ1 positive slides contained a mixture of stained and unstained cells. This variation was seen in both adjacent cells and as an area to area difference. The degree of staining was quantified for p24 and 52K as none, + (50% positive). In this study 2+ or 3+ was regarded as positive and 0 or 1+ as negative. The staining for DF3 antigen was quantified as 0 (none), 1 (pale), 2 (good), 3 (strong), or 4 (intense). For the purpose of this study 3 and 4 were quantified as positive, and 0-2 as negative. Slides with any degree of specific staining with the ER antibody were classified as positive. Mr 24,000protein studies
One hundred-thirteen slides were studied with p24 antibodies of which 10 were technically unevaluable. Of the remaining 103 cases, 63 (61%) were positive and 40 (39%) negative. A statistical correlation between p24 staining and ER was found in the 99 patients for
0
^
10
12
Study year
Fig. 1. Overall Survival (OS) and Disease-Free Survival (DFS) in 103 patients with advanced breast cancer with respect to p24 staining. P-values determined by logrank test.
whom receptor data were available (p - 0.0002) (Table 2). Response (complete and partial remission) to endocrine therapy according to UICC criteria [15] was registered in 43 patients (42%) and no response (no change and progressive disease) in 60 patients (58%). No relationship between response and p24 staining was found (p = 0.37) (Table 3). Despite the significant relationship between p24 and ER no association between p24 and response in the 68 ER-positive patients was found (p = 0.81) (Table 4). Overall survival (p = 0.076) and disease-free survival (p — 0.54) were not related to p24 (Fig. 1). Mr 52,000protein studies
In the 89 slides successfully analyzed, 29 (33%) were positive and 60 (67%) negative. No correlation be-
74 OS
OS 1.0 P = 0.22
0.8 DF3 3-4
1E
\
0.6
DF3 0 - 2 "
0.4 0.2 0.0 100 50
~
0 6
14
8
-^_
^
12
14
10
16
Study year
DFS
DFS 1.0
P = 0.13
0.8
s
1c
0.6
o
a. 8
DF3 3-4 0.4
a.
j
DF3 0-2
^
0.2
•
= ^
1^ -1
0.0
,
i
'
1
100 50 0 6
14
8
10
12
14
Study y«ar
Fig. 2. Overall Survival (OS) and Disease-Free Survival (DFS) in 89 patients with advanced breast cancer with respect to 52K staining. P-values determined by logrank test.
Fig. 3. Overall Survival (OS) and Disease-Free Survival (DFS) in 119 patients with advanced breast cancer with respect to DF3 staining. P-values determined by logrank test.
tween 52K or ER status could be established (p = 0.98) with receptor data available in all patients (Table 2). Response was registered in 38 patients (43%) and nonresponse in 51 patients (57%). The relationship between 52K and response (p = 0.61) is shown in Table 3. The association between response to endocrine therapy and 52K status in 63 patients with ER-positive tumors is given in Table 4 (p = 0.65). Overall survival (p - 0.018) and disease-free survival (p - 0.22) in 52Kpositive and -negative patients is seen in Fig. 2.
A significant correlation (p = 0.044) was established between DF3-positive tumors and ER-positive tumors (Table 2). Response was registered in 45 patients (38%) and non-response (NC+PD) in 74 patients (62%) (p — 0.40) (Table 3). No relationship was found between response to endocrine therapy and DF3 status in 80 patients with ER-positive tumors (p = 0.07) (Table 4). Overall survival (p = 0.22) and disease-free survival (p - 0.13) in DF3-positive and -negative patients is seen in Fig. 3. No inter-relationship between 52K, p24 and DF3 status was found in our material (Table 5).
DF3 studies Estrogen receptor studies
One hundred-nineteen slides were examined for DF3 staining: 44 (37%) of them were positive and 75 (63%) were negative. The same slides were also examined for the presence of ER, which was found in 114 cases.
One hundred-five slides were examined for the presence of ER: 72 (69%) were found to be positive and 33 (31%) negative. Forty 78 14 7 Negative 15 8 7 11 17 10 pmol/mg cytosol protein) and ER status in 242 pre/ 081 p value 0.63 0.S9 0.94 Tumor size perimenopausal women. However, they found no cor>5 cm 17 12 17 11 6 18 22 12 relation between 52K and ER status in postmeno17 28 years 36 22 35 23 14 38 study found overall 52K status uncorrelated to ER
