F. Squartini

Early Pathologic Changes in Experimental and Human Breast Cancer: Facts and Commentsa

All morphologic structures or pathophysiologic changes in the mammary glandular tree develop as a result of the hormonal stimuli that the gland receives. In this respect the breast represents the most typical example in the body of a dynamic structure. In fact, this end organ of the endocrine system converts the hormonal stimuli into morphologic structures, thus almost continuously changing its size, boundaries, and gross and histologic features. This paper deals with the earliest pathologic changes that may be observed in experimental and in human breast cancer, with particular emphasis on the biologic background and progression of these changes. However, only a few points and problems in the biology and pathology of breast carcinogenesis will be raised and commented upon in order to stimulate fruitful speculation and discussion.

p53 Mutations and Histologie Type of Invasive Breast Carcinoma

A polymerase chain reaction-single strand conformation polymorphism assay was used to assess p53 mutations in 148 invasive breast carcinomas, selected on the basis of their histotype. They comprised 56 lobular, 47 ductal, 19 mucinous, 18 medullary, and 8 papillary carcinomas. The distribution of p53 mutations was significantly different (P = 0.006) in the histotypes examined: mutations were frequent in medullary (39%) and ductal (26%), less common in lobular (12%), and absent in mucinous and papillary carcinomas. The frequency of mutations in the exon 5 of the p53 gene was significantly higher in medullary carcinomas than in the other histotypes: 5 (63%) of the mutations found in exon 5 were observed in medullary carcinomas (P = 0.012). One hundred twenty-two tumors from the total were also examined by immunohistochemistry for p53 overexpression using antibody PAb 1801. A specific immunostaining in neoplastic cells was present in 12 tumors. A strong correlation (P < 0.001) was observed between p53 mutations and nuclear accumulation of the p53 protein: 10 tumors were scored positive for both p53 mutation and overexpression. However, in 9 cases having a mutated p53 gene we failed to find a positive immunoreaction. A significant association (P = 0.01) was present between mutations in the p53 gene and high proliferative activity of the tumors determined by immunohistochemistry with monoclonal antibody Ki-67. Moreover, a significantly higher expression of the Ki-67 antigen was found in medullary carcinomas compared to the other histotypes. Our findings indicate that in invasive breast carcinomas structural abnormalities of the p53 gene are mainly seen in medullary and ductal tumors and that the other histological types, especially those associated with a high level of differentiation and favorable prognosis, show a very low incidence of p53 mutations.

Cancerous versus noncancerous breasts: a comparative morphological analysis of the entire glandular tree of the breast

Cancerous and clinically normal autopsy obtained breasts were collected in order to compare the physiopathological profile of both types of glandular tree. Each breast was visualized by whole thin sections and observed under a stereomicroscope with removal of the more interesting changes for histology. The comparison was made between 67 atrophic cancerous breasts and 88 atrophic control breasts. The results were as follows: 25% of the cancerous breasts versus 47% of control breasts showed no changes, atypical lobules, microfoci of “in situ” and/or infiltrating cancer were present in 46% of cancerous breasts and in 16% of control breasts, showing a significant correlation with clinical cancer. All other types of functional and proliferative changes, variously associated each other, were found in 29% of cancerous and in 37% of control breasts. Our morphological data agree completely with the statements in follow-up studies carried out on benign breast biopsies. The significant differences in the physiopathological profile of the glandular tree between “normal” and cancerous breasts, confirms that some changes are causally related to clinical cancer.

MORPHOLOGY AND PROGRESSION OF PRENEOPLASTIC CHANGES IN METHYLCHOLANTHRENE-INDUCED MOUSE MAMMARY TUMORIGENESIS

Susceptible BALB/c virgin female mice were treated with 3-methylcholanthrene in almond oil (0.5 %) by skin painting and killed at monthly intervals. The mammary glands were prepared as whole mounts for stereoscopic study of the glandular tree wtih removal of any focal change for histology. The results showed a marked disarrangement in the architecture of the glandular tree, a prevalent ductal tree, and an overcrowding of ducts. Focal hyperplastic changes were frequently observed, including hyperplastic alveolar nodules, hyperplastic ductal nodules, noduloids and intraductal epithelial growths, and their respective rates of malignant transformation observed at histology were 22 %, 15 %, 7 % and 50 %. Data suggest a different weight of these preneoplastic changes as sources of malignant transformation and a prevalent ductal origin of the methylcholanthrene-induced neoplasms.

Independent submacroscopic foci of infiltrating carcinoma in breasts removed for clinical cancer.

The aim of our study was the detection and the characterization of submacroscopic foci of infiltrating carcinoma in the human female mammary glandular tree collateral to clinical cancer. Accordingly, we analyzed 100 breasts surgically removed by radical mastectomy. Five thin slices per case were analyzed under a dissecting microscope by subgross method of observation. Submacroscopic foci of invasive cancer, well separated and apparently independent of the primary tumor, were found in 19 % of the cases and were confirmed by histologic examination. Foci of submacroscopic cancer were either single (79 %) or multiple (21 %), and were located in slices including or not the main tumor mass (31 % and 69 % of cases, respectively). Their size ranged from 1 to 4 mm. Four histologic types were represented: 1) invasive ductal NOS with productive fibrosis, scirrhous type (36 % of cases); 2) invasive ductal NOS without productive fibrosis, simplex type (32 % of cases); 3) invasive ductal with tubular component (16 % of cases); 4) medullary (16 % of cases). Concordance between histology of clinical and submacroscopic cancers was assessed in 42 % of cases. A significant association of the tubular type (invasive ductal carcinoma with a consistent tubular component) of primary tumor was demonstrated (P < 0.05), as well as with the presence of ductal and lobular proliferative changes in the collateral glandular tree (intraductal papillomas, P < 0.01; atypical lobules, P < 0.02). No relationship was found between submacroscopic foci of infiltrating carcinoma and neoplastic familiarity, patients’ age by decades, axillary lymph node metastases, size of clinical tumor or profile of the collateral mammary glandular tree. These data support the hypothesis of a multicentric origin of human breast cancer and suggest a systemic nature of the neoplastic mammary disease. Prognostic and therapeutic implications of this concept are discussed.

Immunohistochemical analysis of the distribution of a breast tumor associated antigen recognized by a monoclonal antibody

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. MM1-80 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.

FACTORS THAT AFFECT METASTASIS IN VIRUS-INDUCED MOUSE MAMMARY-TUMORS

Two hundred and forty-one mammary tumor-bearing breeding female mice of the BALB/cf C3H and BALB/cfRIII strains, carrying milk-transmitted murine mammary tumor virus (MuMTV) of C3H and RIII origin, respectively, were studied to evaluate the factors that affect tumor metastasis. Only lung metastases were considered and the following factors taken in account: MuMTV inducing variant (C3H, RIII), number of deliveries, tumor histo-type, number of tumors per mouse, clinical duration of tumors, tumor size, and tumor growth rate. Only the number of deliveries, the tumor size and the number of tumors per mouse were found to significantly influence the rate of metastasis. The tumor growth rate affects concurrently with tumor size the metastatic process. In fact, the larger the tumor the higher the tumor growth rate. This direct relationship is significant (P < 0.01) and, in case of lung metastases at autopsy, there was a prevalence of large tumors (> 2 cm) and high growth rate (> 0.3 mm/day). The discordance of these data with those concerning mammary tumor metastasis in virgin females of the same two strains, the enhancing effect of pregnancies on metastatic spread of tumors, and the significance of results for the understanding of the general mechanisms of tumor metastasis are discussed.

Morphologic and antigenic properties of mouse mammary tumor virus produced in a hormone-responsive fashion by C57Bl/10 mammary tumors of non-viral origin.

Bl-MaTU/A1 mouse mammary tumor cells, derived from a C57B1/10 mammary adenocarcinoma induced by dimethylbenzanthracene and mammotropic hormones, express virus particles and proteins related to mouse mammary tumor virus (MMTV). Immunocytochemical analysis by means of monospecific and monoclonal anti-gp52 sera revealed a different localization of the main structural proteins of MMTV in Bl-MaTU/A1 and GR cells (the latter used as a positive virus-producing control). Immunoelectron microscopy of B-type particles budding from the microvilli of dexamethasone-stimulated Bl-MaTU/A1 cells showed remarkably weak reactivity of the viral envelope with anti-gp52-protein A-gold complexes as compared with that of dexamethasone-stimulated GR cells. Since Bl-MaTU/A1-associated MMTV originates from the amplified unit II of endogenous MMTV, which is altered probably within the env gene, the observed antigenic difference in the Bl-MaTU/A1-associated MMTV may be due to altered synthesis of gp52 glycoprotein in these cells.