American Journal of Pathology, Vol. 138, No. 6, June 1991 Copyright ©) American Association of Pathologists
Expression of neu Protein, Epidermal Growth Factor Receptor, and Transforming Growth Factor Alpha in Breast Cancer Correlation with Clinicopathologic Parameters
Joel Lundy,* Allan Schuss,* Doris Stanick,* E. Siobhan McCormack,t Steven Kramer,t and John M. Sorvillot From the Departments of Pathology and Laboratory of
Surgical Oncology, Winthrop Uniersity Hospital, Mineola, and Oncogene Science Inc.,t Manhasset, New York
The major objectives of this study were twofold: to determine 1) if growth factors or growth factor receptors were expressed similarly or differently in a clinically well-characterized group of breast cancer patients and 2) if these phenotypic characteristics were associated with any of the commonly used prognostic parameters. Formalin-fixed paraffinembedded tumor tissue from 51 node-positive breast cancer patients were analyzed for the expression of neu, epidermal growth factor-receptor (EGF-R), and transforming growth factor a (TGFo) using immunoperoxidase staining. Positive membranous stainingfor neu was observed in 15 (29%) tumors. Overexpression of neu was observed in high-grade, estrogen-receptor-negative tumors (P < 0.05). Epidermal growth factor receptor was expressed in 22 (43%) of the tumors analyzed andfound to a greater degree in estrogen-receptor-negative and high-grade tumors (P < 0.025). A significant correlation between neu and EGF-R expression was also noted. Tumors expressing membranous staining of neu had a greater than 70% chance of expressing EGF-R (P < 0.01). Expression of TGFa was found in 68% of tumors and TGFa was detected in grade I and 2 tumor to a greater degree than EGF-R The authors conclude that assaying tumors for these antigens may give additional phenotypic characteristics that can give further insight into the biology of breast cancer. (Am J Pathol 1991,
138:1527-1534)
The neu oncogene is amplified and overexpressed in a number of human adenocarcinoma cell lines.1' 2 The neu oncogene (HER-2 or c-erb 13-2) was first described in association with a neuroblastoma after treatment of rats with a chemical carcinogen.1' 2 The human homolog of the rat neu oncogene has been isolated and encodes for a glycoprotein of 185,000 daltons (P185) that is closely related, but distinct from, the human epidermal growth factor receptor (EGF-R).3'4 Recent results also indicated that neu gene amplification may be associated with early recurrence in node-positive breast cancer and that amplification correlates with elevated levels of expression of neu mRNA and protein.99 The EGF-R is homologous to the c-erb 1-1 oncogene.10 Both EGF-R and neu have a cysteine-rich extracellular domain, a transmembrane domain, and an intracellular tyrosine kinase domain.3 Binding of epidermal growth factor (EGF) to the EGF-R activates a protein kinase, which initiates a mitogenic signal to the cell."1 Epidermal growth factor receptor content in breast cancer tissue has been shown to be inversely related to estrogen receptor content.12 14 Epidermal growth factor receptor expression has been shown also to correlate with other markers of prognosis such as tumor grade.15 Studies have shown that in tumor cell lines expressing both neu and EGF-R, treatment with EGF resulted in phosphorylation of the neu protein; however the ligand for neu has not yet been identified.16 Transforming growth factor-alpha (TGFa) is an acidand heat-stable 50 amino acid protein of 6000 kd molecular weight that also binds to EGF-R and is produced by breast cancer cells.17'18 Transforming growth factoralpha shows 33% amino acid homology with EGF. Both TGFa and EGF bind to the EGF-R receptor, mediate tySupported in part by Oncogene Science Inc. Accepted for publication February 13, 1991. Address reprint requests to Dr. John M. Sorvillo, Oncogene Science Inc., 350 Community Dr., Manhasset, NY 11030.
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AJPJune 1991, Vol. 138, No. 6
rosine phosphorylation of the receptor, and promote anchorage-independent growth of normal rat fibroblasts in soft agar in the presence of transforming growth factor-P.17 Transforming growth factor-alpha is synthesized as a larger precursor of 160 amino acids that is a transmembrane glycoprotein that undergoes proteolytic cleavage releasing TGIa species, ranging in molecular weight from 6 to 27 kd.19 Transforming growth factoralpha secretion can be induced two- to fivefold by estrogens in estrogen-dependent breast cancer cells, and estrogen-independent cells also produce TGFa.18,2021 It has been suggested that TGFa may act as an autocrine growth factor for the proliferation of breast cancer cells. 18,20,21 In recent years, monoclonal antibodies (MAbs) have been used for diagnosis and prognosis of cancer. The aim of our study was to begin to develop a set of phenotypic characteristics for breast cancer based on the expression of neu protein, TGFa, and EGF-R with known clinicopathologic parameters of prognostic value. Formalin-fixed paraffin-embedded tumor tissue sections from 51 breast cancer patients were analyzed for EGF-R, neu, and TGFa expression using MAbs and immunoperoxidase staining. Our results indicate similarities and distinct differences in expression of TGFa, EGF-R, and neu in breast cancer.
Materials and Methods Patient Population Fifty-one node-positive breast cancer cases were selected from the pathology files of Winthrop University Hospital from the year 1988. The age of patients ranged from 39 to 84 years, with a mean age of 61.9 years. Ten patients were premenopausal and 41 postmenopausal. Tumor size (obtained from pathology reports) varied from 0.6 to 7.5 cm. Fourteen patients had Ti tumors, 22 had T2, and 15 had T3. The number of positive nodes per case varied from 1 to 31. Thirty patients had one to three nodes positive and 21 had four or more nodes. Estrogen receptor protein by biochemical determination (more than 10 fmoles/mg cytosol protein was considered positive) was available on 47 of the 51 patients. Thirty-six were estrogen receptor positive (ER) and 25 of these were also progesterone receptor (PgR) positive. Eleven patients were both ER and PgR negative. Tumors were graded histologically in 46 cases according to the criteria of Fisher et al.22 Nineteen tumors were grade 1 or 2, whereas 27 tumors were grade 3.
Monoclonal and Polyclonal Antibodies The MAb c-neu (Ab-2) (Oncogene Science, Inc., Manhasset, NY) was derived by immunization of mice with a synthetic peptide sequence from the kinase domain of human c-neu.8 This antibody, clone 3B5, has been shown previously to specifically immunoprecipitate a 185,000-dalton protein from 35S-methionine-labeled SKBR-3 cells that overexpress neu. The reactivity was shown to be completely blocked by the peptide immunogen.8 The MAb used to evaluate TGFa reactivity, clone 213-4.4, was produced by immunization of mice with recombinant TGFa and has been characterized as previously described.23 This antibody, TGFa (Ab-2) (Oncogene Science, Inc.), reacts with human TGFa and shows no cross-reactivity with EGF. It has been shown to specifically immunoblot mature TGFa and its immunostaining properties have been described.23 Monoclonal antibody to EGF-R, #OM-1 1-951, (Cambridge Research Biochemicals), made against a peptide sequence located in the extracellular domain of the EGF-R, was used in this study. This antibody has been shown to immunoblot 170-kd and 150-kd EGF-R protein from A431 cell extracts that can be specifically competed by immunizing peptide (data provided by manufacturer). A small number of cases were tested with a second antibody, EGF-R (Ab-4) (Oncogene Science, Inc.), which is an affinity-purified polyclonal antibody made to amino acids 1005 through 1016 of the EGF-R. Immunohistochemical staining of EGF-R using either of the antibodies was similar with respect to intensity of staining and the percent positive cells.
Immunohistochemical Staining Formalin-fixed paraffin-embedded tissues sections were used for all studies. Five-micron sections were cut and placed on gelatin-coated slides. Slides were then baked for 45 minutes at 560C. The avidin-biotin complex immunoperoxidase technique was used for all assays.24 Primary antibodies were incubated for 30 minutes at room temperature. The neu antibody was used at a concentration of 0.5 ,ug/ml and the TGFa antibody was used at a concentration of 1 ,ig/mI. The EGF-R MAb (ascites) was used at a 1:2000 dilution with overnight incubation at 40C. Slides were scored in the following fashion: intensity 0 = negative; + = weak staining; + + = moderate staining; + + + = strong staining. Percentage cells positive were assessed in a semiquantitative fashion and divided into the following categories: 0, less than 5%, 5% to 25%, 25% to 50%, and more than 50%. Only membranous staining was considered as positive reactivity using the
Immunohistochemistry, Growth Factors, and Receptors
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AJP June 1991, Vol. 138, No. 6
c-neu (Ab-2) MAb. An arbitrary cutoff based on intensity and percentage cells positive were used to determine positive reactivity to TGF and EGF-R.
Statistical Analysis The chi-square test or Fisher's exact test were used to determine statistical significance of the results.
Results Titration studies of MAbs were performed on frozen and formalin-fixed breast cancer tissue using four concentrations of antibody. The reactivity observed on paraffin sections for these antibodies was equivalent to the reactivity seen using comparable frozen sections. In the case of neu, no background or membranous staining was observed with formalin-fixed benign breast tissue. Both TGFa and EGF reactivity was observed in benign tissues, in agreement with their reported presence in normal breast tissue. Specificity of staining was confirmed by using recombinant TGFa and the synthetic peptide immunogens for EGF-R and neu to abolish reactivity in control tissues (data not shown). A total of 51 cases of nodepositive breast cancer tissue were evaluated for neu and TGFa antigen expression. Only 49 cases were evaluated for EGF-R. The clinicopathologic characteristics of the patient population used in the study are shown in Table 1. Forty-two of the fifty-one cases analyzed exhibited staining of neu protein. Of these 42, 27 showed cytoplasmic staining only, whereas 15 demonstrated membranous staining (Figure 1 A). In 2 of the 15 cases, it was the Table 1. Clinicopathologic Characteristics of 51 Breast Cancer Patients in the Study
Tumor size (cm) 5 Tumor grade Grade 1 and 2 Grade 3 Number of positive nodes 1-3
4-9 >10 Hormone-receptor status (n = 47) ER and PgR positive ER positive only ER and PgR negative n = 51 (all node positive). Age, 39 to 84 years (mean, 61.9 years).
14 22 15
19 27 30 14 7
25 11 11
in situ component (comedo-type) only that demonstrated membranous staining. In the remaining 13 cases in which the membranous reactivity was seen in the invasive component, 12 showed similar staining with respect to the intensity and percentage of cells positive in the regional nodes (Figure 1 B). Analysis of neu membrane staining in relation to the clinicopathologic characteristics of the patient population is shown in Table 2. Seven of thirty cases (23%) with one to three positive nodes expressed membranous staining of neu, whereas 8 of 21 (38%) with four or more positive nodes showed membranous staining. Only 3 of 14 (21 %) of Ti tumors (less than 2 cm) demonstrated membranous reactivity, whereas 12 of 37 (32%) of larger tumors showed membranous staining. Interestingly 12 of the 13 invasive cancers with membranous staining were grade 3 tumors. Seven of eleven ER-negative tumors demonstrated membranous staining and only 6 of 36 ERpositive tumors (P < 0.01). (Hormone receptor data was not available on four tumors.) Tumors from 3 of 10 premenopausal patients and 12 of 41 postmenopausal patients showed membranous reactivity. Table 3 shows the analysis of the same patient population using TGFa MAbs. Thirty-five of fifty-one cases analyzed showed positive staining for TGFa. Interestingly less reactivity was noted in regional nodes compared with primary tumors (Figure 2A, B). There was little difference in reactivity seen based on tumor size, number of positive nodes, ER status, or menopausal status. Analysis of the same patient population using EGF-R MAbs is shown in Table 4. A trend was noted for increased EGF-R expression in larger tumors that were ER negative (Figure 3). This is in agreement with previous reports that have shown that EGF-R expression in breast tumors is inversely proportional to estrogen receptor content.12"14 Only 5 of 19 (26%) grade 1 and 2 tumors expressed EGF-R, whereas 16 of 26 (62%) grade 3 tumors were positive (P < 0.025). A comparison of differences between neu, EGF-R, and TGFot expression is summarized in Table 5. Ti tumors (less than 2 cm) showed greater expression of TGFa than neu. Interestingly no such difference was seen with larger tumors (more than 2 cm). Greater TGFa expression also was seen in the patients with one to three nodes. Although reactivity was similar in ER-negative cases, TGFa expression was greater in ER-positive cases. In the 13 cases that could be graded with membranous staining, 12 were grade 3. In nine of these cases, TGFa expression was less than neu expression, in three they were the same, and they were greater in only 1. In the 45 cases graded, only 3 of 19 (16%) grade 1 and 2 cases were not reactive to anti-TGFa, but 11 of 26 (41%) grade 3 cases were nonreactive (Table 5). Trans-
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Figure 1. A: A primary poorly differentiated infiltrating duct carcinoma demonstrating the membranous staining pattern of the neu. Arrow indicates stained cells (X400). B: Regional lymph node corresponding to the above primary tumor showing tumor cells with a similar neu membranous staining pattern. Arrow indicates stained cells (X400).
Table 2. neu Membrane Staining Versus Clinicopathologic Parameters of 51 Patients No. of patients Characteristic Membrane staining Present Absent (n = 15) (n = 36) Tumor size (cm) 2 12 (32%) 25 (68%) Nodal status 1-3 nodes 7 (23%) 23 (77%) >4 nodes 8 (38%) 13 (62%) Estrogen receptor Positive 6 (17%) 30 (83%) Negative 7 (64%) 4 (36%) Menopausal status Premenopausal 3 (0.3%) 7 (0.7%) Postmenopausal 12 (29%) 29 (71%)
Total 51 14 37
30 21 36 11 10 41
Twelve of thirteen cases in which the invasive component expressed membranous staining were grade III (poorly differentiated).
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AJPJune 1991, Vol. 138, No. 6
Table 3. TGFa Staining Versus Clinicopathologic Paramneters of 51 Patients No. of Patients
35)
Absent (n = 16)
Total 51
11 (79%) 24 (65%)
13 (35%)
3(21%)
14 37
22 (73%) 13 (62%)
8 (27%) 8 (38%)
30 21
25 (69%) 6 (55%)
11 (30%) 5 (45%)
36 11
6 (60%) 29 (71%)
4 (40%) 12 (29%)
10 41
Present
Characteristics
(n Tumor size (cm) 2 Nodal status 1-3 nodes >4 nodes Estrogen receptor Positive Negative Menopausal status Premenopausal Postmenopausal
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Expression of neu Protein, Epidermal Growth Factor Receptor, and Transforming Growth Factor Alpha in Breast Cancer Correlation with Clinicopathologic Parameters
Joel Lundy,* Allan Schuss,* Doris Stanick,* E. Siobhan McCormack,t Steven Kramer,t and John M. Sorvillot From the Departments of Pathology and Laboratory of
Surgical Oncology, Winthrop Uniersity Hospital, Mineola, and Oncogene Science Inc.,t Manhasset, New York
The major objectives of this study were twofold: to determine 1) if growth factors or growth factor receptors were expressed similarly or differently in a clinically well-characterized group of breast cancer patients and 2) if these phenotypic characteristics were associated with any of the commonly used prognostic parameters. Formalin-fixed paraffinembedded tumor tissue from 51 node-positive breast cancer patients were analyzed for the expression of neu, epidermal growth factor-receptor (EGF-R), and transforming growth factor a (TGFo) using immunoperoxidase staining. Positive membranous stainingfor neu was observed in 15 (29%) tumors. Overexpression of neu was observed in high-grade, estrogen-receptor-negative tumors (P < 0.05). Epidermal growth factor receptor was expressed in 22 (43%) of the tumors analyzed andfound to a greater degree in estrogen-receptor-negative and high-grade tumors (P < 0.025). A significant correlation between neu and EGF-R expression was also noted. Tumors expressing membranous staining of neu had a greater than 70% chance of expressing EGF-R (P < 0.01). Expression of TGFa was found in 68% of tumors and TGFa was detected in grade I and 2 tumor to a greater degree than EGF-R The authors conclude that assaying tumors for these antigens may give additional phenotypic characteristics that can give further insight into the biology of breast cancer. (Am J Pathol 1991,
138:1527-1534)
The neu oncogene is amplified and overexpressed in a number of human adenocarcinoma cell lines.1' 2 The neu oncogene (HER-2 or c-erb 13-2) was first described in association with a neuroblastoma after treatment of rats with a chemical carcinogen.1' 2 The human homolog of the rat neu oncogene has been isolated and encodes for a glycoprotein of 185,000 daltons (P185) that is closely related, but distinct from, the human epidermal growth factor receptor (EGF-R).3'4 Recent results also indicated that neu gene amplification may be associated with early recurrence in node-positive breast cancer and that amplification correlates with elevated levels of expression of neu mRNA and protein.99 The EGF-R is homologous to the c-erb 1-1 oncogene.10 Both EGF-R and neu have a cysteine-rich extracellular domain, a transmembrane domain, and an intracellular tyrosine kinase domain.3 Binding of epidermal growth factor (EGF) to the EGF-R activates a protein kinase, which initiates a mitogenic signal to the cell."1 Epidermal growth factor receptor content in breast cancer tissue has been shown to be inversely related to estrogen receptor content.12 14 Epidermal growth factor receptor expression has been shown also to correlate with other markers of prognosis such as tumor grade.15 Studies have shown that in tumor cell lines expressing both neu and EGF-R, treatment with EGF resulted in phosphorylation of the neu protein; however the ligand for neu has not yet been identified.16 Transforming growth factor-alpha (TGFa) is an acidand heat-stable 50 amino acid protein of 6000 kd molecular weight that also binds to EGF-R and is produced by breast cancer cells.17'18 Transforming growth factoralpha shows 33% amino acid homology with EGF. Both TGFa and EGF bind to the EGF-R receptor, mediate tySupported in part by Oncogene Science Inc. Accepted for publication February 13, 1991. Address reprint requests to Dr. John M. Sorvillo, Oncogene Science Inc., 350 Community Dr., Manhasset, NY 11030.
1527
1528
Lundy et al
AJPJune 1991, Vol. 138, No. 6
rosine phosphorylation of the receptor, and promote anchorage-independent growth of normal rat fibroblasts in soft agar in the presence of transforming growth factor-P.17 Transforming growth factor-alpha is synthesized as a larger precursor of 160 amino acids that is a transmembrane glycoprotein that undergoes proteolytic cleavage releasing TGIa species, ranging in molecular weight from 6 to 27 kd.19 Transforming growth factoralpha secretion can be induced two- to fivefold by estrogens in estrogen-dependent breast cancer cells, and estrogen-independent cells also produce TGFa.18,2021 It has been suggested that TGFa may act as an autocrine growth factor for the proliferation of breast cancer cells. 18,20,21 In recent years, monoclonal antibodies (MAbs) have been used for diagnosis and prognosis of cancer. The aim of our study was to begin to develop a set of phenotypic characteristics for breast cancer based on the expression of neu protein, TGFa, and EGF-R with known clinicopathologic parameters of prognostic value. Formalin-fixed paraffin-embedded tumor tissue sections from 51 breast cancer patients were analyzed for EGF-R, neu, and TGFa expression using MAbs and immunoperoxidase staining. Our results indicate similarities and distinct differences in expression of TGFa, EGF-R, and neu in breast cancer.
Materials and Methods Patient Population Fifty-one node-positive breast cancer cases were selected from the pathology files of Winthrop University Hospital from the year 1988. The age of patients ranged from 39 to 84 years, with a mean age of 61.9 years. Ten patients were premenopausal and 41 postmenopausal. Tumor size (obtained from pathology reports) varied from 0.6 to 7.5 cm. Fourteen patients had Ti tumors, 22 had T2, and 15 had T3. The number of positive nodes per case varied from 1 to 31. Thirty patients had one to three nodes positive and 21 had four or more nodes. Estrogen receptor protein by biochemical determination (more than 10 fmoles/mg cytosol protein was considered positive) was available on 47 of the 51 patients. Thirty-six were estrogen receptor positive (ER) and 25 of these were also progesterone receptor (PgR) positive. Eleven patients were both ER and PgR negative. Tumors were graded histologically in 46 cases according to the criteria of Fisher et al.22 Nineteen tumors were grade 1 or 2, whereas 27 tumors were grade 3.
Monoclonal and Polyclonal Antibodies The MAb c-neu (Ab-2) (Oncogene Science, Inc., Manhasset, NY) was derived by immunization of mice with a synthetic peptide sequence from the kinase domain of human c-neu.8 This antibody, clone 3B5, has been shown previously to specifically immunoprecipitate a 185,000-dalton protein from 35S-methionine-labeled SKBR-3 cells that overexpress neu. The reactivity was shown to be completely blocked by the peptide immunogen.8 The MAb used to evaluate TGFa reactivity, clone 213-4.4, was produced by immunization of mice with recombinant TGFa and has been characterized as previously described.23 This antibody, TGFa (Ab-2) (Oncogene Science, Inc.), reacts with human TGFa and shows no cross-reactivity with EGF. It has been shown to specifically immunoblot mature TGFa and its immunostaining properties have been described.23 Monoclonal antibody to EGF-R, #OM-1 1-951, (Cambridge Research Biochemicals), made against a peptide sequence located in the extracellular domain of the EGF-R, was used in this study. This antibody has been shown to immunoblot 170-kd and 150-kd EGF-R protein from A431 cell extracts that can be specifically competed by immunizing peptide (data provided by manufacturer). A small number of cases were tested with a second antibody, EGF-R (Ab-4) (Oncogene Science, Inc.), which is an affinity-purified polyclonal antibody made to amino acids 1005 through 1016 of the EGF-R. Immunohistochemical staining of EGF-R using either of the antibodies was similar with respect to intensity of staining and the percent positive cells.
Immunohistochemical Staining Formalin-fixed paraffin-embedded tissues sections were used for all studies. Five-micron sections were cut and placed on gelatin-coated slides. Slides were then baked for 45 minutes at 560C. The avidin-biotin complex immunoperoxidase technique was used for all assays.24 Primary antibodies were incubated for 30 minutes at room temperature. The neu antibody was used at a concentration of 0.5 ,ug/ml and the TGFa antibody was used at a concentration of 1 ,ig/mI. The EGF-R MAb (ascites) was used at a 1:2000 dilution with overnight incubation at 40C. Slides were scored in the following fashion: intensity 0 = negative; + = weak staining; + + = moderate staining; + + + = strong staining. Percentage cells positive were assessed in a semiquantitative fashion and divided into the following categories: 0, less than 5%, 5% to 25%, 25% to 50%, and more than 50%. Only membranous staining was considered as positive reactivity using the
Immunohistochemistry, Growth Factors, and Receptors
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AJP June 1991, Vol. 138, No. 6
c-neu (Ab-2) MAb. An arbitrary cutoff based on intensity and percentage cells positive were used to determine positive reactivity to TGF and EGF-R.
Statistical Analysis The chi-square test or Fisher's exact test were used to determine statistical significance of the results.
Results Titration studies of MAbs were performed on frozen and formalin-fixed breast cancer tissue using four concentrations of antibody. The reactivity observed on paraffin sections for these antibodies was equivalent to the reactivity seen using comparable frozen sections. In the case of neu, no background or membranous staining was observed with formalin-fixed benign breast tissue. Both TGFa and EGF reactivity was observed in benign tissues, in agreement with their reported presence in normal breast tissue. Specificity of staining was confirmed by using recombinant TGFa and the synthetic peptide immunogens for EGF-R and neu to abolish reactivity in control tissues (data not shown). A total of 51 cases of nodepositive breast cancer tissue were evaluated for neu and TGFa antigen expression. Only 49 cases were evaluated for EGF-R. The clinicopathologic characteristics of the patient population used in the study are shown in Table 1. Forty-two of the fifty-one cases analyzed exhibited staining of neu protein. Of these 42, 27 showed cytoplasmic staining only, whereas 15 demonstrated membranous staining (Figure 1 A). In 2 of the 15 cases, it was the Table 1. Clinicopathologic Characteristics of 51 Breast Cancer Patients in the Study
Tumor size (cm) 5 Tumor grade Grade 1 and 2 Grade 3 Number of positive nodes 1-3
4-9 >10 Hormone-receptor status (n = 47) ER and PgR positive ER positive only ER and PgR negative n = 51 (all node positive). Age, 39 to 84 years (mean, 61.9 years).
14 22 15
19 27 30 14 7
25 11 11
in situ component (comedo-type) only that demonstrated membranous staining. In the remaining 13 cases in which the membranous reactivity was seen in the invasive component, 12 showed similar staining with respect to the intensity and percentage of cells positive in the regional nodes (Figure 1 B). Analysis of neu membrane staining in relation to the clinicopathologic characteristics of the patient population is shown in Table 2. Seven of thirty cases (23%) with one to three positive nodes expressed membranous staining of neu, whereas 8 of 21 (38%) with four or more positive nodes showed membranous staining. Only 3 of 14 (21 %) of Ti tumors (less than 2 cm) demonstrated membranous reactivity, whereas 12 of 37 (32%) of larger tumors showed membranous staining. Interestingly 12 of the 13 invasive cancers with membranous staining were grade 3 tumors. Seven of eleven ER-negative tumors demonstrated membranous staining and only 6 of 36 ERpositive tumors (P < 0.01). (Hormone receptor data was not available on four tumors.) Tumors from 3 of 10 premenopausal patients and 12 of 41 postmenopausal patients showed membranous reactivity. Table 3 shows the analysis of the same patient population using TGFa MAbs. Thirty-five of fifty-one cases analyzed showed positive staining for TGFa. Interestingly less reactivity was noted in regional nodes compared with primary tumors (Figure 2A, B). There was little difference in reactivity seen based on tumor size, number of positive nodes, ER status, or menopausal status. Analysis of the same patient population using EGF-R MAbs is shown in Table 4. A trend was noted for increased EGF-R expression in larger tumors that were ER negative (Figure 3). This is in agreement with previous reports that have shown that EGF-R expression in breast tumors is inversely proportional to estrogen receptor content.12"14 Only 5 of 19 (26%) grade 1 and 2 tumors expressed EGF-R, whereas 16 of 26 (62%) grade 3 tumors were positive (P < 0.025). A comparison of differences between neu, EGF-R, and TGFot expression is summarized in Table 5. Ti tumors (less than 2 cm) showed greater expression of TGFa than neu. Interestingly no such difference was seen with larger tumors (more than 2 cm). Greater TGFa expression also was seen in the patients with one to three nodes. Although reactivity was similar in ER-negative cases, TGFa expression was greater in ER-positive cases. In the 13 cases that could be graded with membranous staining, 12 were grade 3. In nine of these cases, TGFa expression was less than neu expression, in three they were the same, and they were greater in only 1. In the 45 cases graded, only 3 of 19 (16%) grade 1 and 2 cases were not reactive to anti-TGFa, but 11 of 26 (41%) grade 3 cases were nonreactive (Table 5). Trans-
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_J ,sNwae
AP June 1991, Vol. 138, No. 6
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Figure 1. A: A primary poorly differentiated infiltrating duct carcinoma demonstrating the membranous staining pattern of the neu. Arrow indicates stained cells (X400). B: Regional lymph node corresponding to the above primary tumor showing tumor cells with a similar neu membranous staining pattern. Arrow indicates stained cells (X400).
Table 2. neu Membrane Staining Versus Clinicopathologic Parameters of 51 Patients No. of patients Characteristic Membrane staining Present Absent (n = 15) (n = 36) Tumor size (cm) 2 12 (32%) 25 (68%) Nodal status 1-3 nodes 7 (23%) 23 (77%) >4 nodes 8 (38%) 13 (62%) Estrogen receptor Positive 6 (17%) 30 (83%) Negative 7 (64%) 4 (36%) Menopausal status Premenopausal 3 (0.3%) 7 (0.7%) Postmenopausal 12 (29%) 29 (71%)
Total 51 14 37
30 21 36 11 10 41
Twelve of thirteen cases in which the invasive component expressed membranous staining were grade III (poorly differentiated).
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Table 3. TGFa Staining Versus Clinicopathologic Paramneters of 51 Patients No. of Patients
35)
Absent (n = 16)
Total 51
11 (79%) 24 (65%)
13 (35%)
3(21%)
14 37
22 (73%) 13 (62%)
8 (27%) 8 (38%)
30 21
25 (69%) 6 (55%)
11 (30%) 5 (45%)
36 11
6 (60%) 29 (71%)
4 (40%) 12 (29%)
10 41
Present
Characteristics
(n Tumor size (cm) 2 Nodal status 1-3 nodes >4 nodes Estrogen receptor Positive Negative Menopausal status Premenopausal Postmenopausal
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