Breast CancerResearch and Treatment15: 213-216, 1990. © 1990KluwerAcademic Publishers. Printedin the Netherlands. Brief communication
Tissue carcinoembryonic antigen in the prognosis of early invasive breast cancer
Roberto Hegg, 1 Aurelio Z. De Souza, 1 Cynthia Bomfim Pestana 2 and Paulo C. Cardoso De Almeida 3 1Department of Gynecology, Hospital das Clinicas, University of S~o Paulo School of Medicine; 2Department of Pathology, Instituto Adolfo Lutz; ~Division of Anatomical Pathology, Hospital das Clinicas, University of S(to Paulo School of Medicine
Key words: breast cancer, carcinoembryonic antigen (CEA), nodal status, survival rate Summary Twenty-five patients with stage II ductal breast carcinoma followed up for ten years were studied for the presence of tissue carcinoembryonic antigen (CEA). Overall expression of CEA was 60%. The ten year survival rate was significantly higher for patients with CEA-negative turnouts (70%) than for patients with CEA-positive tumours (27%), while the difference between the survival rate of patients with (30%) or without (53%) lymph node involvement did not reach significance. Among the 10 patients with lymph node involvement, CEA-negative patients had a better outcome. These results suggest that there is a correlation between the presence of tissue CEA and the prognosis of the disease, and that CEA status might possibly be more important than lymph node involvement, at least within stage II breast carcinomas.
Introduction Prognosis for breast cancer patients is based on several parameters such as patient age, growth rate, site of primary tumours, local extension, subcutaneous lymphatic permeation, histological type and grade, hormonal status, tumour size, and lymph node metastases. The axillary lymph node status and tumour size have been used as the best prognostic indicators [1]. The lymph node positive patients demonstrate 25% disease-free survival at ten years compared to 75% for lymph node negative mastectomy patients [2]. The latter show an approximate 5 % annual recurrence rate over a ten year period [3]. The usefulness of carcinoembryonic antigen (CEA) as a marker in patients with breast carcinoma has been established in many studies. However,
studies using the immunoperoxidase technique to detect CEA in histologic sections of breast cancer have provided conflicting results. There are some publications in which CEA immunoreactivity in breast cancer was found to be correlated with a worse prognosis [4-8] whereas other authors [9-12] failed to find this correlation. None of these studies have had a population of patients with a well-defined clinical stage and long-term follow-up. The aim of the present study was to correlate the long-term follow-up of early invasive breast cancer (stage II TNM-UICC) with the presence of tissue CEA.
Materials and methods A study was carried out on twenty five patients
Addressfor offprints: P.C. Cardosode Almeida,Rua ConselheiroBrotero, 1505;CEP 01232- SiloPaulo-SP- Brasil
214
R Hegg et al.
with stage II breast ductal carcinoma (TNMUICC) aged 31 to 69 years (mean age 49 years) treated by radical mastectomy and complementary radiotherapy and followed up for at least 10 years. For the purpose of this study the surviving rate was defined by the percentage of patients who were alive with no evidence of disease after ten years from the first surgery. Twelve patients (48%) were in menacme and thirteen (52%) were in menopause. Ten of these patients (40%) had lymph node metastases. None of them had other previous malignancies or autoimmune diseases. Immunoperoxidase studies were performed on deparaffinized sections of formalin-fixed tissue using Sternberger's peroxidase-antiperoxidase technique [13]. Polyclonal antisera against CEA (Dakopatts, Santa Barbara, CA) were employed. Briefly, histological sections were hydrated, endogenous peroxidase was blocked with 0.3% H202 and non-specific binding with 10% normal goat serum, and the material was incubated with the primary antibody 1 : 300 for 30 minutes at room temperature. Sections were then incubated consecutively with goat anti-rabbit IgG for 30 minutes and rabbit peroxidase-antiperoxidase complex (Dakopatts). Thorough washing with PBS (phosphate buffered saline), pH 7.6, was carried out between the various stages. The substrate used was 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sigma). Sections were counterstained with hematoxylin, dehydrated, and mounted in balsam. Data were analysed by the chi-square contingency tables. Probabilities were calculated with one degree of freedom.
Results
Strong intracytoplasmic staining was observed in most of the neoplastic cells from 15 CEA-positive tumours (60%). Table I presents the survival rates according to the presence of CEA in the primary tumour and the lymph node status. There was a significant correlation between CEA positivity and lower survival rates during a ten year follow-up (p < 0.05). There was not a significant difference in survival rates between cases with positive (30%) and negative (53%) lymph nodes. The relationship between axillary lymph node status and presence or absence of CEA is shown in Table 2. The presence of CEA immunoreactivity in the primary tumour showed no significant correlation with the presence of lymph node metastases (p < 0.25). Twenty per cent of the CEA negative turnouts had metastases and 80% had no metastases. Of the CEA-positive turnouts, 53 % had metastases and 47% had none. Prognosis seemed to be worst for patients presenting both CEA positivity and lymph node involvement; for the cases with lymph node metastases the survival rates were 12% and 100% for CEA-positive and CEA-negative respectively. Although the number of cases is too small to confirm this difference statistically, these rates suggest that patients with CEA-negative tumours might have better prognosis than patients with CEA-positive tumours, regardless of axillary lymph node involvement.
Discussion
The presence of CEA has been associated with several tumours suggesting a more aggressive beTable 1. Ten year survival rate according to CEA in the primary tumor and lymph node (LN) status CEA positive No patients No surviving
Table 2. Ten year survival rate according to combined lymph node (LN) and CEA status in the primary tumour
LN negative
positive
negative
15 (60%) 10 (40%) 10 (40%) 15 (60%) 4 (27%)* 7 (70%)* 3 (30%) + 8 (53%) +
* Significance of difference: p < 0.05. + Significance of difference: p < 0.25.
LN and CEA status
No of patients
No surviving
LNLNLN + LN+
8 7 2 8
5 (62%) 3 (43%) 2 (100%) 1 (12%)
CEACEA + CEA CEA+
Tissue carcinoembryonic antigen
havior [14]. The incidence of CEA positivity in breast carcinomas investigated immunohistochemically has ranged from 1.6% in one study [15] to 42-83% in others [4-8, 10-12]. In our study 60% of the turnouts were CEA positive, in keeping with most of other reported series. Possible explanations for such a wide variation may be the differences in the antisera used as well as staining methods, purification, and scoring for positivity. The correlation between CEA immunoreactivity and poor prognosis is not well established. The few studies performed to demonstrate such correlation have yielded conflicting results. Shousha and Lyssiotis [4] observed a correlation between CEA positivity in the primary tumour and the presence of lymph node metastases, a parameter that has been frequently used as a prognostic indicator. Such a correlation was not significant in the present small study and others [10-12]. Kuhadja et al. [7] only observed this relationship when tumours smaller than 3 cm were considered. The influence of size on metastatic potential may explain the discrepant conclusions concerning CEA positivity and metastases in the literature, as the positive correlation between increasing size of primary breast carcinoma and lymph node metastases has long been recognized [16]. Shousha et al. [5] in another study with patients followed up for a period of 6 to 13 years found that the 5 and 10 year survival rates were significantly higher in patients with CEA-negative tumours and that this relation was independent of the presence of lymph node metastases. Mansour et al. [8] also observed that prognosis correlated better with CEA status than with lymph node or estrogen receptor status. Our results are consistent with most of these conclusions. The survival rate over a ten year follow-up was significantly higher for patients with CEA-negative tumours (70%) than for patients with CEA-positive tumours (27%), suggesting that a relationship between the presence of CEA in histological sections of stage II breast carcinoma and the prognosis of the disease does exist. This correlation does not seem to depend on axillary lymph node involvement. The correlation between the survival rate of patients with axillary lymph node involvement (30%) and without axil-
215
lary lymph node involvement (53 %) was not significant in this study, and among the patients with lymph node involvement, the survival rate was higher for patients with CEA-negative tumours (100%) than for patients with CEA-positive tumours (12%) in our study. Other results, however, have also been published. The correlation between CEA positivity and recurrence within 2 years of the discovery of primary disease was not observed by Walter [10]. Possible explanations for these different results are the short follow-up period of the patients investigated and different stages of tumours analyzed. Halter et al. [11] did not find a relationship between expression of CEA and patient prognosis in either regional or localized breast carcinoma, nor did Van Der Linden et al. [12] who did not correlate CEA positivity with established prognostic factors (clinical, histopathological, morphometric, and biochemical features). Histological types, stage of tumours, and patient follow-up were not mentioned in the latter study. This difference of results may be related to the clinical stage of the tumours, which is not mentioned in several studies. If many cases of high stage tumours are included in such studies, final low survival rates may interfere with the results. It is generally accepted that axillary lymph node involvement is the most relevant factor in establishing the prognosis for breast carcinoma. The present study considers only Stage II tumours, thus excluding node-negative tumours if they are small (Stage I), so that the influence of nodal status alone as a predictor of outcome cannot be fairly tested. Nevertheless, according to our study, the presence of tissue CEA in Stage II tumours may be more important than axillary involvement in these tumours, and may be a helpful indicator of worse prognosis. This finding is based on a small number of cases and should be confirmed by other authors.
References 1. FisherB: Laboratory and clinic research in breast cancer. A personal adventure. Cancer Res 40:3863-3874, 1980 2. Fisher B, Slack N, Katrych D, Wolmark N: Ten year fol-
216
3.
4.
5.
6.
7.
8.
9.
R Hegg et al.
low-up results of patients with carcinoma of the breast in a cooperative clinical trial evaluating surgical adjuvant chemotherapy. Surg Gynecol Obstet 140: 528--534, 1975 Albano WA, Hanf CD, Organ CH: Natural history of lymph node-negative breast cancer. Surgery 86: 574-577, 1979 Shousha S, Lyssiotis T: Correlation of carcinoembryonic antigen in tissue sections with spread of mammary carcinoma. Histopathology 2: 443-447, 1978 Shousha S, Lyssiotis T, Godfrey VM, Schever PJ: Carcinoernbryonic antigen in breast cancer tissue: a useful prognostic indicator. Br Med J 1: 777-779, 1979 Wharen B, Lidbrink E, Wallgren A, Eneroth P, Zajicek J: Carcinoembryonic antigen and other tumour markers in tissue and serum or plasma of patients with primary mammary carcinoma. Cancer 42: 1870--1878, 1978 Kuhadja FJ, Offutt LE, Mendelsohn G: The distribution of carcinoembryonic antigen in breast carcinoma. Cancer 52: 1257-1264, 1978 Mansour GE, Hasert M, Park CH, Koehler KA, Petrelli M: Tissue and plasma carcinoembryonic antigen in early breast cancer. Cancer 51: 1243-1248, 1983 Persijn JP, Korsten CB: Carcinoembryonic antigens, oes-
10.
11.
12.
13.
14. 15.
16.
trogen receptors and androgen receptors in human breast tumours. J Clin Chem Clin Biochem 15: 553-556, 1980 Walker RA: Demonstration of carcinoembryonic antigen in human breast carcinomas by the immunoperoxidase technique. J Clin Pathol 33: 356-360, 1980 Halter SA, Fraker LD, Parmenter M, Dupont WD: Carcinoembryonic antigen expression and patient survival in carcinoma of the breast. Oncology 41: 297-302, 1984 Van Der Linden JC, Baak JP, Lindeman J, Smeulders AWM, Meyer CJL: Carcinoembryonic antigen expression and peanut agglutinin binding in clinical, histopathological, biochemical and morphometric features. Histopathology 9: 1051-1059, 1985 Sternberger LA: The unlabeled antibody peroxidase antiperoxidase (PAP) method, ln: Wiley J (ed) Immunohistochemistry. New York, 1979, pp 104-168 Uriel J: Retrodifferentiation and the fetal patterns of gene expression in cancer. Adv Cancer Res 29: 127-174, 1979 Goldenberg DM, Sharkey RM, Primus FJP: Immunohistochemical detection of carcinoembryonic antigen in conventional histopathology specimens. Cancer 42: 1546-1553, 1978 Donegan W, Spratt J: Cancer of the Breast (2nd ed). W.B. Saunders, Philadelphia, 1979, p 86
Tissue carcinoembryonic antigen in the prognosis of early invasive breast cancer
Roberto Hegg, 1 Aurelio Z. De Souza, 1 Cynthia Bomfim Pestana 2 and Paulo C. Cardoso De Almeida 3 1Department of Gynecology, Hospital das Clinicas, University of S~o Paulo School of Medicine; 2Department of Pathology, Instituto Adolfo Lutz; ~Division of Anatomical Pathology, Hospital das Clinicas, University of S(to Paulo School of Medicine
Key words: breast cancer, carcinoembryonic antigen (CEA), nodal status, survival rate Summary Twenty-five patients with stage II ductal breast carcinoma followed up for ten years were studied for the presence of tissue carcinoembryonic antigen (CEA). Overall expression of CEA was 60%. The ten year survival rate was significantly higher for patients with CEA-negative turnouts (70%) than for patients with CEA-positive tumours (27%), while the difference between the survival rate of patients with (30%) or without (53%) lymph node involvement did not reach significance. Among the 10 patients with lymph node involvement, CEA-negative patients had a better outcome. These results suggest that there is a correlation between the presence of tissue CEA and the prognosis of the disease, and that CEA status might possibly be more important than lymph node involvement, at least within stage II breast carcinomas.
Introduction Prognosis for breast cancer patients is based on several parameters such as patient age, growth rate, site of primary tumours, local extension, subcutaneous lymphatic permeation, histological type and grade, hormonal status, tumour size, and lymph node metastases. The axillary lymph node status and tumour size have been used as the best prognostic indicators [1]. The lymph node positive patients demonstrate 25% disease-free survival at ten years compared to 75% for lymph node negative mastectomy patients [2]. The latter show an approximate 5 % annual recurrence rate over a ten year period [3]. The usefulness of carcinoembryonic antigen (CEA) as a marker in patients with breast carcinoma has been established in many studies. However,
studies using the immunoperoxidase technique to detect CEA in histologic sections of breast cancer have provided conflicting results. There are some publications in which CEA immunoreactivity in breast cancer was found to be correlated with a worse prognosis [4-8] whereas other authors [9-12] failed to find this correlation. None of these studies have had a population of patients with a well-defined clinical stage and long-term follow-up. The aim of the present study was to correlate the long-term follow-up of early invasive breast cancer (stage II TNM-UICC) with the presence of tissue CEA.
Materials and methods A study was carried out on twenty five patients
Addressfor offprints: P.C. Cardosode Almeida,Rua ConselheiroBrotero, 1505;CEP 01232- SiloPaulo-SP- Brasil
214
R Hegg et al.
with stage II breast ductal carcinoma (TNMUICC) aged 31 to 69 years (mean age 49 years) treated by radical mastectomy and complementary radiotherapy and followed up for at least 10 years. For the purpose of this study the surviving rate was defined by the percentage of patients who were alive with no evidence of disease after ten years from the first surgery. Twelve patients (48%) were in menacme and thirteen (52%) were in menopause. Ten of these patients (40%) had lymph node metastases. None of them had other previous malignancies or autoimmune diseases. Immunoperoxidase studies were performed on deparaffinized sections of formalin-fixed tissue using Sternberger's peroxidase-antiperoxidase technique [13]. Polyclonal antisera against CEA (Dakopatts, Santa Barbara, CA) were employed. Briefly, histological sections were hydrated, endogenous peroxidase was blocked with 0.3% H202 and non-specific binding with 10% normal goat serum, and the material was incubated with the primary antibody 1 : 300 for 30 minutes at room temperature. Sections were then incubated consecutively with goat anti-rabbit IgG for 30 minutes and rabbit peroxidase-antiperoxidase complex (Dakopatts). Thorough washing with PBS (phosphate buffered saline), pH 7.6, was carried out between the various stages. The substrate used was 0.05% 3,3'-diaminobenzidine tetrahydrochloride (Sigma). Sections were counterstained with hematoxylin, dehydrated, and mounted in balsam. Data were analysed by the chi-square contingency tables. Probabilities were calculated with one degree of freedom.
Results
Strong intracytoplasmic staining was observed in most of the neoplastic cells from 15 CEA-positive tumours (60%). Table I presents the survival rates according to the presence of CEA in the primary tumour and the lymph node status. There was a significant correlation between CEA positivity and lower survival rates during a ten year follow-up (p < 0.05). There was not a significant difference in survival rates between cases with positive (30%) and negative (53%) lymph nodes. The relationship between axillary lymph node status and presence or absence of CEA is shown in Table 2. The presence of CEA immunoreactivity in the primary tumour showed no significant correlation with the presence of lymph node metastases (p < 0.25). Twenty per cent of the CEA negative turnouts had metastases and 80% had no metastases. Of the CEA-positive turnouts, 53 % had metastases and 47% had none. Prognosis seemed to be worst for patients presenting both CEA positivity and lymph node involvement; for the cases with lymph node metastases the survival rates were 12% and 100% for CEA-positive and CEA-negative respectively. Although the number of cases is too small to confirm this difference statistically, these rates suggest that patients with CEA-negative tumours might have better prognosis than patients with CEA-positive tumours, regardless of axillary lymph node involvement.
Discussion
The presence of CEA has been associated with several tumours suggesting a more aggressive beTable 1. Ten year survival rate according to CEA in the primary tumor and lymph node (LN) status CEA positive No patients No surviving
Table 2. Ten year survival rate according to combined lymph node (LN) and CEA status in the primary tumour
LN negative
positive
negative
15 (60%) 10 (40%) 10 (40%) 15 (60%) 4 (27%)* 7 (70%)* 3 (30%) + 8 (53%) +
* Significance of difference: p < 0.05. + Significance of difference: p < 0.25.
LN and CEA status
No of patients
No surviving
LNLNLN + LN+
8 7 2 8
5 (62%) 3 (43%) 2 (100%) 1 (12%)
CEACEA + CEA CEA+
Tissue carcinoembryonic antigen
havior [14]. The incidence of CEA positivity in breast carcinomas investigated immunohistochemically has ranged from 1.6% in one study [15] to 42-83% in others [4-8, 10-12]. In our study 60% of the turnouts were CEA positive, in keeping with most of other reported series. Possible explanations for such a wide variation may be the differences in the antisera used as well as staining methods, purification, and scoring for positivity. The correlation between CEA immunoreactivity and poor prognosis is not well established. The few studies performed to demonstrate such correlation have yielded conflicting results. Shousha and Lyssiotis [4] observed a correlation between CEA positivity in the primary tumour and the presence of lymph node metastases, a parameter that has been frequently used as a prognostic indicator. Such a correlation was not significant in the present small study and others [10-12]. Kuhadja et al. [7] only observed this relationship when tumours smaller than 3 cm were considered. The influence of size on metastatic potential may explain the discrepant conclusions concerning CEA positivity and metastases in the literature, as the positive correlation between increasing size of primary breast carcinoma and lymph node metastases has long been recognized [16]. Shousha et al. [5] in another study with patients followed up for a period of 6 to 13 years found that the 5 and 10 year survival rates were significantly higher in patients with CEA-negative tumours and that this relation was independent of the presence of lymph node metastases. Mansour et al. [8] also observed that prognosis correlated better with CEA status than with lymph node or estrogen receptor status. Our results are consistent with most of these conclusions. The survival rate over a ten year follow-up was significantly higher for patients with CEA-negative tumours (70%) than for patients with CEA-positive tumours (27%), suggesting that a relationship between the presence of CEA in histological sections of stage II breast carcinoma and the prognosis of the disease does exist. This correlation does not seem to depend on axillary lymph node involvement. The correlation between the survival rate of patients with axillary lymph node involvement (30%) and without axil-
215
lary lymph node involvement (53 %) was not significant in this study, and among the patients with lymph node involvement, the survival rate was higher for patients with CEA-negative tumours (100%) than for patients with CEA-positive tumours (12%) in our study. Other results, however, have also been published. The correlation between CEA positivity and recurrence within 2 years of the discovery of primary disease was not observed by Walter [10]. Possible explanations for these different results are the short follow-up period of the patients investigated and different stages of tumours analyzed. Halter et al. [11] did not find a relationship between expression of CEA and patient prognosis in either regional or localized breast carcinoma, nor did Van Der Linden et al. [12] who did not correlate CEA positivity with established prognostic factors (clinical, histopathological, morphometric, and biochemical features). Histological types, stage of tumours, and patient follow-up were not mentioned in the latter study. This difference of results may be related to the clinical stage of the tumours, which is not mentioned in several studies. If many cases of high stage tumours are included in such studies, final low survival rates may interfere with the results. It is generally accepted that axillary lymph node involvement is the most relevant factor in establishing the prognosis for breast carcinoma. The present study considers only Stage II tumours, thus excluding node-negative tumours if they are small (Stage I), so that the influence of nodal status alone as a predictor of outcome cannot be fairly tested. Nevertheless, according to our study, the presence of tissue CEA in Stage II tumours may be more important than axillary involvement in these tumours, and may be a helpful indicator of worse prognosis. This finding is based on a small number of cases and should be confirmed by other authors.
References 1. FisherB: Laboratory and clinic research in breast cancer. A personal adventure. Cancer Res 40:3863-3874, 1980 2. Fisher B, Slack N, Katrych D, Wolmark N: Ten year fol-
216
3.
4.
5.
6.
7.
8.
9.
R Hegg et al.
low-up results of patients with carcinoma of the breast in a cooperative clinical trial evaluating surgical adjuvant chemotherapy. Surg Gynecol Obstet 140: 528--534, 1975 Albano WA, Hanf CD, Organ CH: Natural history of lymph node-negative breast cancer. Surgery 86: 574-577, 1979 Shousha S, Lyssiotis T: Correlation of carcinoembryonic antigen in tissue sections with spread of mammary carcinoma. Histopathology 2: 443-447, 1978 Shousha S, Lyssiotis T, Godfrey VM, Schever PJ: Carcinoernbryonic antigen in breast cancer tissue: a useful prognostic indicator. Br Med J 1: 777-779, 1979 Wharen B, Lidbrink E, Wallgren A, Eneroth P, Zajicek J: Carcinoembryonic antigen and other tumour markers in tissue and serum or plasma of patients with primary mammary carcinoma. Cancer 42: 1870--1878, 1978 Kuhadja FJ, Offutt LE, Mendelsohn G: The distribution of carcinoembryonic antigen in breast carcinoma. Cancer 52: 1257-1264, 1978 Mansour GE, Hasert M, Park CH, Koehler KA, Petrelli M: Tissue and plasma carcinoembryonic antigen in early breast cancer. Cancer 51: 1243-1248, 1983 Persijn JP, Korsten CB: Carcinoembryonic antigens, oes-
10.
11.
12.
13.
14. 15.
16.
trogen receptors and androgen receptors in human breast tumours. J Clin Chem Clin Biochem 15: 553-556, 1980 Walker RA: Demonstration of carcinoembryonic antigen in human breast carcinomas by the immunoperoxidase technique. J Clin Pathol 33: 356-360, 1980 Halter SA, Fraker LD, Parmenter M, Dupont WD: Carcinoembryonic antigen expression and patient survival in carcinoma of the breast. Oncology 41: 297-302, 1984 Van Der Linden JC, Baak JP, Lindeman J, Smeulders AWM, Meyer CJL: Carcinoembryonic antigen expression and peanut agglutinin binding in clinical, histopathological, biochemical and morphometric features. Histopathology 9: 1051-1059, 1985 Sternberger LA: The unlabeled antibody peroxidase antiperoxidase (PAP) method, ln: Wiley J (ed) Immunohistochemistry. New York, 1979, pp 104-168 Uriel J: Retrodifferentiation and the fetal patterns of gene expression in cancer. Adv Cancer Res 29: 127-174, 1979 Goldenberg DM, Sharkey RM, Primus FJP: Immunohistochemical detection of carcinoembryonic antigen in conventional histopathology specimens. Cancer 42: 1546-1553, 1978 Donegan W, Spratt J: Cancer of the Breast (2nd ed). W.B. Saunders, Philadelphia, 1979, p 86
