Path. Res. Pract. 188, 1002-1008 (1992)
Immunohistochemical Analysis of the Distribution of a Breast Tumor Associated Antigen Recognized by a Monoclonal Antibody M. Castagnal, P. Viacaval, V. Turchi 2 , A. Rughetti 2 , M. Nuti 2 and F. SquartinP llnstitute of Pathological Anatomy, University of Pisa, Pisa; 2Department of Experimental Medicine, Rome, Italy
SUMMARY A new monoclonal antibody (MoAb), MM 1-80, recognizing a tumor associated epitope of a breast high molecular weight mucin molecule was tested, using the avidin biotin immunoperoxidase method on normal and pathological mammary tissues. The normal mammary ducts and lobules were negative. Fibroadenomas showed a strong intracytoplasmic staining. In apocrine metaplasia, adenosis, and papillomatosis, scattered cells showed intracytoplasmic, luminal border or secretion reactivity. In lobular and ductal hyperplasia the cells showed intracytoplasmic immunoreactivity which, however, became more intense and homogeneous in atypical lesions, i.e. lobular and ductal in-situ carcinomas. The infiltrating carcinomas of different histotype expressed positivity on 98 % of the cases (113/115) and axillary metastatic lymph nodes were always positive (20/20). The MoAb was tested on 175 human neoplasias of different origin which were in the majority of the cases negative with the exception of adenocarcinomas of the lung, ovary and bladder. MMl-80 appears to react preferentially with mammary cells undergoing hyperplastic, metaplastic and neoplastic processes. The 1-80 epitope distribution is different in these lesions starting with a predominant luminal expression in benign lesions and becoming strong and cytoplasmic in the malignant breast cell.
Introduction In the last few years there has been a remarkable production of MoAbs against tumor-associated antigens of different human neoplasias. In many instances, however, the produced antibodies show a variable range of reactiviry recognizing only some of the carcinomas and within the tumor a portion of the cancer cells 11 , 18. Several of the MoAbs that have been generated to react against breast carcinoma cells are now known to recognize different epitopes of the PEM (Polymorphic Epithelial Mucin)2,4, 7, 10, 15, 17. 0344-0338/92/0188-1002$3.50/0
Since this molecule is produced by normal and tumor epithelial cells from different anatomical sites, it is important to define the MoAbs overall reactivity using immunohistochemical methods. In fact it appears for some of the MoAbs raised against these mucins that the recognized epitopes are tumor-associated since they represent "newly" exposed antigenic determinants due to aberrant glycosilation that can be totally masked in normal cells 7,8. . In this study we have analyzed the reactivity of one of these MoAbs, MMl-80, on normal and pathologic mammary tissues and on tissues of other origin. The antibody appears of relevant interest for the immunohistopathological application in the differential diagnosis of ©
1992 by
Gustav Fischer Verlag, Stuttgart
Monoclonal Antibody in Breast Cancer· 1003
occult breast carcinoma. Previous studies!4 during the characterization of the antibody have shown a selective reactivity with breast carcinoma cells by indirect immunofluorescence on frozen sections and ELISA. Moreover, it has become obvious in the studies that the corresponding epitope is nbt affected by formalin fixation and paraffin embedding.
Material and Methods The production and characterization of the monoclonal antibody MMI-80 has been described elsewhere 14 . The MoAb is of the IgM isotype. Briefly Balblc mice were immunized with a pool of membrane-enriched fractions of lymph nodal metastases of human mammary cancer obtained from different patients. Tissue samples removed at surgery for breast pathological lesions performed at the Institute of Pathological Anatomy of the University of Pisa were fixed in 10 % formalin and embedded in paraffin. Hematoxylin-eosin sections were examined in order to select different lesions. The hyperplastic lesions were classified in epitheliosis and atypical lobular and ductal hyperplasias 1, 15, 19. The tumors were divided according to the current classification of mammary carcinomas. Additional 5 ~M sections of the selected blocks were tested with MoAb MMl-80 using the immunoperoxidase-avidin method (Vector). For the evaluation and comparison of the results the following criteria were used: a) number of positive cases; b) site of positivity, whether intracytoplasmic, luminal cell border or luminal secretion; c) number of positive cells per case expressed by percentage (10-50% or 50-100%); In this study the MoAb MMl-80 ascitic fluid was used at a 1: 500 dilution and the incubation period was at 4°C in a humid chamber overnight. In every experiment a negative control such as omitting the primary antibody or using a non-related antibody of identical isotype was performed.
Results The reactivity of the MoAb MMl-80 was assessed on normal mammary lobules and ducts, fibroadenomas, papillomas, adenosis, apocrine metaplasia, ductal and lobular hyperplastic lesions, lobular and ductal in-situ carcinomas, infiltrating carcinomas of different histotype with and without metastatic axillary lymph nodes and metastatic axillary lymph nodes for a total number of 270 cases. Table 1 shows the summary of the results concerning the benign and in-situ neoplastic breast lesions. The cells of the normal ducts and lobules always resulted to be negative. Fibroadenomas (10 cases) were all positive and showed a strong intracytoplasmic positivity. Adenosis 43 presented-, a different pattern of antigen distribution with staining in the cytoplasm of scattered luminal cell border.
Table 1. Immunoperoxidase reactivity of MoAb MMl-80 with normal structures, fibroadenomas, hyperplastic and metaplastic lesions and in-situ carcinoma of the human breast Analyzed structures
Number of positivel number tested
Normal ducts and lobules
0/31
Fibroadenomas Adenosis Apocrine MetaplaSla Papillomas Epitheliosis Atypical lobular hyperplasia Atypical ductal hyperplasia Lobular in-situ carcmomas Ductal in-situ carcmomas
10/10 43/43 22/22
Total
717 12112 2/2
Antigen distribution
secretion border cytoplasm
10 4
35 22
10 15 4
3
7 9 2
12 2
7
7
717
SIS
5
27/27
27
135/135
17
83
82
The luminal border of all 22 apocrine metaplasias tested was labeled with MoAb MMl-80; four of these lesions also showed a cytoplasmic immunostaining. A total of 12 epithelioses, 2 atypical lobular and 7 atypical ductal hyperplasias were tested and all of them resulted positive with intracytoplasmatic staining. This type of antigen distribution was also found in 5 lobular and 27 ductal in~situ carcinomas. In eight cases of epitheliosis the number of positive cells was higher than 50 %; in four cases it was lower. Two cases of atypical lobular hyperplasia showed heterogeneous staining with a number of reactive cells lower than 50 %. In lobular carcinomas the reactivity was mainly heterogeneous (4 cases out of 5) and the number of positive cells lower than 50 %. Fig. 1 panel illustrates a case of epitheliosis with stained scattered cells. The atypical ductal hyperplasia (Fig. 1, panel b) showed a percentage of reactive cells which was higher than 50 % in 7/7 cases with homogeneous staining of atypical cells. All the in-situ duct carcinomas were positive (27127) and 21 of these showed a high number of positive cells. Table 2 reports the summary of the MMl-80 immunoperoxidase reactivity on a total of 115 breast carcinomas with 5 ! and without 64 axillary metastatic lymph nodes at surgery. No differences could be observed between the two sets of primary tumors (N + and N -) in terms of MMl-80 antigenic expression. The MoAb MMl-80 reactivity included all different his to types corresponding to 96 % (49/51) in N+ and 100 % (64/64) in N-.
Monoclonal Antibody in Breast Cancer· 1005 Table 2. Immunoperoxidase reactivity of MoAb MMl-80 on primary breast cancers with and without axillary metastatic lymph nodes Tumor Histotype
Number of Antigen Distribution positiveltotal number tested cytoplasm membrane
- Infiltrating ductal carcinoma not otherwise specified - Infiltrating lobular carcinoma - Mucinous carcinoma - Medullary carcinoma - Tubular carcinoma - Papillary carcinoma - Paget carcinoma of nipple - Other carcinomas total
N-
N+
N-
N+
N-
N+
35/35
28/29
33
28
5
4
9/9 3/3 10/10 2/2 3/3 2/2
6/6 2/2 6/7 4/4
9 1 10 2 1 2
6 1 6 4
0 2 1
3 2 1
64/64
Total
3/3 49/51
58
113/115
Table 3. Immunoperoxidase reactivity of monoclonal antibody MM1-S0 on 20 formalin fixed primary breast tumors and autologous metastatic lymph nodes Primary breast tumors percent cell staining 100 90 SO 70 60 50 40 30 20 10
6 4 1
Metastatic axillary lymph nodes
11 1
2 3
2 2 2
1
1
2
1
1
37/49 for N+ and 62164 for N - of the positive tumors showed positive cells in more than 50 % of the total number of cancer cells present in the specimen. The staining pattern in positive tumors was usually cytoplasmic diffuse (48/49 in N + and 58/64 in N -) and sometimes extended to the cell membrane (10/49 in N+ and 10164 in N-). Table 3 shows in more detail the distribution of the MMl-80 antigen on 20 breast cancer cases with the relative autologous lymph nodal metastases considering the percentage of tumor cells positive in stained sections.
Immunohistochemical Analysis of the Distribution of a Breast Tumor Associated Antigen Recognized by a Monoclonal Antibody M. Castagnal, P. Viacaval, V. Turchi 2 , A. Rughetti 2 , M. Nuti 2 and F. SquartinP llnstitute of Pathological Anatomy, University of Pisa, Pisa; 2Department of Experimental Medicine, Rome, Italy
SUMMARY A new monoclonal antibody (MoAb), MM 1-80, recognizing a tumor associated epitope of a breast high molecular weight mucin molecule was tested, using the avidin biotin immunoperoxidase method on normal and pathological mammary tissues. The normal mammary ducts and lobules were negative. Fibroadenomas showed a strong intracytoplasmic staining. In apocrine metaplasia, adenosis, and papillomatosis, scattered cells showed intracytoplasmic, luminal border or secretion reactivity. In lobular and ductal hyperplasia the cells showed intracytoplasmic immunoreactivity which, however, became more intense and homogeneous in atypical lesions, i.e. lobular and ductal in-situ carcinomas. The infiltrating carcinomas of different histotype expressed positivity on 98 % of the cases (113/115) and axillary metastatic lymph nodes were always positive (20/20). The MoAb was tested on 175 human neoplasias of different origin which were in the majority of the cases negative with the exception of adenocarcinomas of the lung, ovary and bladder. MMl-80 appears to react preferentially with mammary cells undergoing hyperplastic, metaplastic and neoplastic processes. The 1-80 epitope distribution is different in these lesions starting with a predominant luminal expression in benign lesions and becoming strong and cytoplasmic in the malignant breast cell.
Introduction In the last few years there has been a remarkable production of MoAbs against tumor-associated antigens of different human neoplasias. In many instances, however, the produced antibodies show a variable range of reactiviry recognizing only some of the carcinomas and within the tumor a portion of the cancer cells 11 , 18. Several of the MoAbs that have been generated to react against breast carcinoma cells are now known to recognize different epitopes of the PEM (Polymorphic Epithelial Mucin)2,4, 7, 10, 15, 17. 0344-0338/92/0188-1002$3.50/0
Since this molecule is produced by normal and tumor epithelial cells from different anatomical sites, it is important to define the MoAbs overall reactivity using immunohistochemical methods. In fact it appears for some of the MoAbs raised against these mucins that the recognized epitopes are tumor-associated since they represent "newly" exposed antigenic determinants due to aberrant glycosilation that can be totally masked in normal cells 7,8. . In this study we have analyzed the reactivity of one of these MoAbs, MMl-80, on normal and pathologic mammary tissues and on tissues of other origin. The antibody appears of relevant interest for the immunohistopathological application in the differential diagnosis of ©
1992 by
Gustav Fischer Verlag, Stuttgart
Monoclonal Antibody in Breast Cancer· 1003
occult breast carcinoma. Previous studies!4 during the characterization of the antibody have shown a selective reactivity with breast carcinoma cells by indirect immunofluorescence on frozen sections and ELISA. Moreover, it has become obvious in the studies that the corresponding epitope is nbt affected by formalin fixation and paraffin embedding.
Material and Methods The production and characterization of the monoclonal antibody MMI-80 has been described elsewhere 14 . The MoAb is of the IgM isotype. Briefly Balblc mice were immunized with a pool of membrane-enriched fractions of lymph nodal metastases of human mammary cancer obtained from different patients. Tissue samples removed at surgery for breast pathological lesions performed at the Institute of Pathological Anatomy of the University of Pisa were fixed in 10 % formalin and embedded in paraffin. Hematoxylin-eosin sections were examined in order to select different lesions. The hyperplastic lesions were classified in epitheliosis and atypical lobular and ductal hyperplasias 1, 15, 19. The tumors were divided according to the current classification of mammary carcinomas. Additional 5 ~M sections of the selected blocks were tested with MoAb MMl-80 using the immunoperoxidase-avidin method (Vector). For the evaluation and comparison of the results the following criteria were used: a) number of positive cases; b) site of positivity, whether intracytoplasmic, luminal cell border or luminal secretion; c) number of positive cells per case expressed by percentage (10-50% or 50-100%); In this study the MoAb MMl-80 ascitic fluid was used at a 1: 500 dilution and the incubation period was at 4°C in a humid chamber overnight. In every experiment a negative control such as omitting the primary antibody or using a non-related antibody of identical isotype was performed.
Results The reactivity of the MoAb MMl-80 was assessed on normal mammary lobules and ducts, fibroadenomas, papillomas, adenosis, apocrine metaplasia, ductal and lobular hyperplastic lesions, lobular and ductal in-situ carcinomas, infiltrating carcinomas of different histotype with and without metastatic axillary lymph nodes and metastatic axillary lymph nodes for a total number of 270 cases. Table 1 shows the summary of the results concerning the benign and in-situ neoplastic breast lesions. The cells of the normal ducts and lobules always resulted to be negative. Fibroadenomas (10 cases) were all positive and showed a strong intracytoplasmic positivity. Adenosis 43 presented-, a different pattern of antigen distribution with staining in the cytoplasm of scattered luminal cell border.
Table 1. Immunoperoxidase reactivity of MoAb MMl-80 with normal structures, fibroadenomas, hyperplastic and metaplastic lesions and in-situ carcinoma of the human breast Analyzed structures
Number of positivel number tested
Normal ducts and lobules
0/31
Fibroadenomas Adenosis Apocrine MetaplaSla Papillomas Epitheliosis Atypical lobular hyperplasia Atypical ductal hyperplasia Lobular in-situ carcmomas Ductal in-situ carcmomas
10/10 43/43 22/22
Total
717 12112 2/2
Antigen distribution
secretion border cytoplasm
10 4
35 22
10 15 4
3
7 9 2
12 2
7
7
717
SIS
5
27/27
27
135/135
17
83
82
The luminal border of all 22 apocrine metaplasias tested was labeled with MoAb MMl-80; four of these lesions also showed a cytoplasmic immunostaining. A total of 12 epithelioses, 2 atypical lobular and 7 atypical ductal hyperplasias were tested and all of them resulted positive with intracytoplasmatic staining. This type of antigen distribution was also found in 5 lobular and 27 ductal in~situ carcinomas. In eight cases of epitheliosis the number of positive cells was higher than 50 %; in four cases it was lower. Two cases of atypical lobular hyperplasia showed heterogeneous staining with a number of reactive cells lower than 50 %. In lobular carcinomas the reactivity was mainly heterogeneous (4 cases out of 5) and the number of positive cells lower than 50 %. Fig. 1 panel illustrates a case of epitheliosis with stained scattered cells. The atypical ductal hyperplasia (Fig. 1, panel b) showed a percentage of reactive cells which was higher than 50 % in 7/7 cases with homogeneous staining of atypical cells. All the in-situ duct carcinomas were positive (27127) and 21 of these showed a high number of positive cells. Table 2 reports the summary of the MMl-80 immunoperoxidase reactivity on a total of 115 breast carcinomas with 5 ! and without 64 axillary metastatic lymph nodes at surgery. No differences could be observed between the two sets of primary tumors (N + and N -) in terms of MMl-80 antigenic expression. The MoAb MMl-80 reactivity included all different his to types corresponding to 96 % (49/51) in N+ and 100 % (64/64) in N-.
Monoclonal Antibody in Breast Cancer· 1005 Table 2. Immunoperoxidase reactivity of MoAb MMl-80 on primary breast cancers with and without axillary metastatic lymph nodes Tumor Histotype
Number of Antigen Distribution positiveltotal number tested cytoplasm membrane
- Infiltrating ductal carcinoma not otherwise specified - Infiltrating lobular carcinoma - Mucinous carcinoma - Medullary carcinoma - Tubular carcinoma - Papillary carcinoma - Paget carcinoma of nipple - Other carcinomas total
N-
N+
N-
N+
N-
N+
35/35
28/29
33
28
5
4
9/9 3/3 10/10 2/2 3/3 2/2
6/6 2/2 6/7 4/4
9 1 10 2 1 2
6 1 6 4
0 2 1
3 2 1
64/64
Total
3/3 49/51
58
113/115
Table 3. Immunoperoxidase reactivity of monoclonal antibody MM1-S0 on 20 formalin fixed primary breast tumors and autologous metastatic lymph nodes Primary breast tumors percent cell staining 100 90 SO 70 60 50 40 30 20 10
6 4 1
Metastatic axillary lymph nodes
11 1
2 3
2 2 2
1
1
2
1
1
37/49 for N+ and 62164 for N - of the positive tumors showed positive cells in more than 50 % of the total number of cancer cells present in the specimen. The staining pattern in positive tumors was usually cytoplasmic diffuse (48/49 in N + and 58/64 in N -) and sometimes extended to the cell membrane (10/49 in N+ and 10164 in N-). Table 3 shows in more detail the distribution of the MMl-80 antigen on 20 breast cancer cases with the relative autologous lymph nodal metastases considering the percentage of tumor cells positive in stained sections.
