Bioactivity of C3H and RIII mammary tumor viruses in virgin female BALB/c mice.

The bioactivities of C3H and RIII mammary tumor virus (MuMTV) in virgin female BALB/c mice differed. The average number of mammary hyperplastic alveolar nodules per mouse after noduligenic tests was 20.1 in BALB/cfC3H and 10.7 in BALB/cfRIII females. Spontaneous mammary tumor incidence after 20 months of observation was 47.5% in BALB/cfC3H and 14.6% in BALB/cfRIII females (P less than 0.01). The frequency of lung metastases in mammary tumor-bearing mice was 63.1% in BALB/cfC3H and 16.6% in BALB/cfRIII females (P less than 0.05), although the clinical duration of mammary tumors was the same. These data demonstrated a lower bioactivity of RIII MuMTV when compared to C3H MuMTV in BALB/c mice and suggested that the causative virus may control all the steps of mouse mammary tumor development, including metastasis.

Development, morphology, and progression of mammary tumors during and after fertile life in BALB/cfRIII mice.

A study was done on 368 BALB/cfRIII mammary tumor-bearing breeding female mice that are of the BALB/c genotype and carry milk-transmitted RIII murine mammary tumor virus (MuMTV) infection initiated by foster-nursing. All tumors were characterized by chronologic, biologic, and morphologic analyses. Mammary tumors occurring during pregnancy-lactation periods were mostly pregnancy-dependent plaques showing organoid tubular structure, sudden growth during late pregnancy, regression after delivery, and slow progression. The frequency of pregnancy-dependent tumors per 100 mice at risk increased steadily from first (1 tumor) to sixth (38 tumors) pregnancy-lactation period. Mammary tumors occurring after fertile life were mostly characterized by an irregular, slow growth and a type B morphology. The frequency of these tumors per 100 mice at risk per 100 days of survival also increased regularly in females undergoing from one (73 tumors) to six (117 tumors) pregnancy-lactation periods. Data were discussed in the light of the pattern of MuMTV release through BALB/cfRIII milk.

Differences in progression of BALB/cfRIII and BALB/cfC3H mammary hyperplastic alveolar nodules transplanted into the gland-free fat pads of BALB/c mice.

In BALB/cfC3H and BALB/cfRIII virgin female mice, i.e., BALB/c mice carrying milk-transmitted C3H and those carrying RIII murine mammary tumor virus infection, respectively, mammary noduligenesis and tumorigenesis are remarkably different. Focal hyperplastic alveolar changes or hyperplastic alveolar nodules were removed from BALB/cfRIII or BALB/cfC3H breeding female donors and transplanted into the gland-free mammary fat pads of syngeneic BALB/c virgin female recipients with or without hormonal supplementation. The results obtained have shown significant differences in: (a) the morphological type of outgrowths; (b) the incidence of malignant transformations; and (c) the hormonal responsiveness of the tumors arisen after pituitary transplants.

Mammary cancer antigen recognized by monoclonal antibody B72.3 in apocrine metaplasia of the human breast.

Monoclonal antibody B72.3 recognizing a pan-associated carcinoma antigen expressed also in metastatic human breast cancer cells has been tested using the avidin-biotin peroxidase method applied to paraffin-embedded sections in 50 samples of mammary tissue showing apocrine metaplasia and in 58 cases of other mild or severe focal epithelial proliferative changes of the breast, including mostly in situ lobular or ductal carcinomas collateral to clinical cancer removed after radical mastectomy. The antigen detected by this antibody was present in the apocrine cells of 48 cases (96%). In the majority of these cases the reactivity was localized on the luminal border of the apocrine cells and in the luminal secretion. But ten cases showed positive staining also in the cell cytoplasm either focal or diffuse. The normal structures and mild focal hyperplastic changes collateral to clinical cancer were, in the majority of the cases (43 of 55), negative, and, when positive, displayed positivity only at the luminal border. By contrast, the independent foci of in situ carcinoma (17 of 31 examined), the intraduct papillomas (seven cases of 14), and the intraductal component of breast carcinoma (seven cases of 17) were positive, displaying a cytoplasmic focal or diffuse staining. In conclusion, mammary apocrine metaplasia, a metaplastic change of the normal epithelium that has been associated with increased breast cancer risk, shares antigens in common with breast cancer cells and/or with cells showing severe atypia. The possible clinical significance of the site of antigenic expression (cytoplasm or luminal border) needs further investigation.

Lobuloalveolar differentiation and tumorigenesis: two separate activities of mouse mammary tumor virus.

The morphological growth of the mammary glandular tree from 3 to 18 months of age has been quantified in three groups of genetically identical virgin female mice: BALB/c mice free of milk-transmitted mouse mammary tumor virus (MMTV) infection and BALB/cfC3H and BALB/cfRIII mice carrying milk-transmitted MMTV infection of C3H or RIII origin, respectively. Mice were killed at 3-month intervals, and their mammary glands were prepared for stereoscopic examination as whole mounts. Any cluster of alveoli from 0.1 mm in diameter was counted and measured at 0.1-mm intervals. The small clusters of alveoli (less than or equal to 0.3 mm) were used to measure the amount of lobuloalveolar differentiation. The larger clusters of alveoli, the hyperplastic alveolar nodules, and the mammary tumors were used to measure the frequency of tumor cell transformations. Lobuloalveolar differentiation in BALB/c mice was increased significantly by both C3H (p less than 0.001) and RIII (p less than 0.001) MMTV infections. RIII MMTV induced a significantly higher lobuloalveolar differentiation as compared to that induced by C3H MMTV (p less than 0.001). C3H MMTV showed a significantly higher capability in inducing hyperplastic alveolar nodules and mammary tumors as compared to that of RIII MMTV (p less than 0.001). The conclusions were that two different activities are carried out by MMTV, namely, lobuloalveolar differentiation and noduligenesis, and that these two MMTV activities are distinct from each other.

Reciprocal interference between milk-transmitted mammary tumor virus and Friend leukemia viruses in mice: possible role of the interferon system.

We have previously shown that mice simultaneously infected with the murine mammary tumor virus (MuMTV) and with certain slow murine leukemia viruses (MLV) have an increased resistance to the pathological effects of both agents. Here we report that milk-transmitted MuMTV also delays the development of the acute erythroleukemia induced by Friend leukemia virus (FLV), and retards, or prevents in some cases, the development of long-term lymphomas caused by its helper component. This is confirmed by the observation that the average life span of MuMTV-carrying mice infected with FLV or its helper component is prolonged by over 30% as compared to that of MuMTV-free animals infected with the same agents and by the finding that the replication of Friend viruses is reduced in mice neonatally exposed to MuMTV. Since the antibody responses of mice to MuMTV and to FLV were not cross-reactive, we searched for antiviral activity in the tissues of mice exposed to MuMTV, FLV, or the helper component of FLV. Low levels of interferon-alpha/beta were consistently detected in spleen extracts from mice infected with all these agents but could not be demonstrated in the spleens of uninfected BALB/c mice; thus, the chronic production of endogenous interferon is likely to play a major role in the reciprocal interference in vivo between retroviruses belonging to different genera.

Comparison of monoclonal immunocytochemical and immunoenzymatic methods for steroid receptor evaluation in breast cancer.

The production of monoclonal antibodies against estrogen receptor (ER) and progesterone receptor (PR) has permitted the development of the enzyme immunoassay (EIA) and immunocytochemical assay (ICA) for steroid receptor determination. The results obtained with these two techniques, using the same monoclonal antibodies, were compared in a large series of breast carcinomas (187 for ER and 100 for PR). The correlation between these methods was significant for ER (rs = 0.54) and PR (rs = 0.55) (P less than 0.001) but was lost when the receptor concentrations determined by EIA were less than or equal to 15 and less than or equal to 30 fmol/mg protein for ER and PR, respectively. When these values are considered as cutoffs, the concordance between the two methods was 84.5% for ER and 73% for PR. An analysis of discordant results revealed that low epithelial cellularity generally was present in ICA-positive, EIA-negative specimens, whereas only focal positivity with ICA, or positivity of only normal peripheral mammary ducts and lobules, frequently was found in ICA-negative, EIA-positive tumors. In conclusion, there is good correlation between the results obtained by EIA and ICA methods for detection of ER and PR. The authors suggest that biochemical and histochemical methods for steroid receptors could be considered complementary and used together for the analysis of breast cancer.

Correlation between 5-year survival of T1-T2N0 breast cancer and some pathological parameters.

In order to more clearly define the factors influencing prognosis, 113 cases of T1-T2N0 breast cancers, all operated on by means of radical mastectomy, were reviewed. The following pathologic parameters were analyzed in relation to 5 years survival: histological type, nuclear grading, vascular embolization, size and border of the tumour, and sinus histiocytosis, diffuse cortical hyperplasia and germinal center hyperplasia of axillary nodes. We observed a close correlation between the presence of sinus histiocytosis and prognosis, as many authors have reported. Diffuse cortical hyperplasia stands out from our data as the most unfavourable prognostic factor.

p53 expression in non small cell lung cancer: clinical and biological correlations.

p53 is a tumor suppressor gene, located in the short arm of chromosome 17, which encodes for a nuclear protein involved in the control of cellular growth. Mutations in p53 gene are the most common genetic alterations in a several human cancers, including Non Small Cell Lung Cancer (NSCLC). However, up to now, the role of p53 in the tumour's behaviour and its progression has not been completely clear. We performed immunohistochemical staining for mutated p53 using two monoclonal antibodies, PAb1801 and PAb240, in fresh tumour specimens from 103 consecutive patients who underwent surgery for resectable NSCLC. PAb1801 detects both the normal and mutant form of p53, while PAb240 is specific only for the mutant form and recognizes a denaturation-resistant epitope located between aminoacids 156-335. Both antibodies showed a mainly nuclear staining in neoplastic cells but not in surrounding uninvolved lung tissues. 68 out of 100 (68%) and 37 out of 103 (35.9%) of the cases were positive with PAb1801 and with PAb240, respectively. Tumours from patients with hilar-mediastinal lymph node involvement showed a higher p53 expression, detected by PAb1801, than those without nodal metastases (p = 0.04). Moreover, tumours expressing more than 60% of positive cells with both antibodies showed a significant increase of nodal involvement (p = 0.1; p = 0.03). Furthermore, p53 expression was significantly related to post-surgical stage (p Tumor Stage) (p = 0.04). In addition, we did not find any correlation between p53 expression and proliferating activity evaluated by PCNA, Ki-67 and DNA flow cytometric cell cycle. In conclusion, the evaluation of p53 oncogene expression may identify individuals whose resectable NSCLCs have a more aggressive tumour behaviour.

Estrogen, progesterone receptors and proliferating activity evaluated by immunocytochemistry in breast cancer.

The correlation of the most important prognostic indicators was evaluated in 75 breast cancer cases. Estrogen-progesterone receptors and proliferating activity were analyzed by immunocytochemical methods (ER-ICA, PR-ICA, Ki-67). Both steroid receptors were inversely correlated with the proliferating activity (ER-ICA vs Ki-67, p less than 0.003; PR-ICA vs. Ki-67, p less than 0.0001). No correlation was found between steroid receptors or cell kinetics and tumor size or lymph node status. These findings confirm the relevance of biochemical and kinetic parameters as independent markers in breast cancer and suggest a routine use of the simple immunocytochemical methods in assessing the biological behavior of tumors.

Value of ER-D5 immunocytochemical determination in routine tissue sections of human breast cancer.

ER-D5 is a recently identified protein related to estrogen receptors (ER). Generally ER measurement requires fresh frozen tissue and for ER-D5 assay ethanol (E) fixation of the specimen is recommended. We evaluated the possibility of immunocytochemical detection of ER-D5 in routine formalin-fixed (F) sections in 51 breast cancers comparing the results with those obtained in the same specimens using E as fixative. The results of ER-D5 assay were expressed by the staining index (SI) taking values greater than or equal to 5 as positive. In all tumors ER was also assayed by a biochemical method (DCCA). The sensitivity of ER-D5 detection in F was only 33.3%, while the specificity was 94.4%. A lower cut-off value of SI for F sections (greater than or equal to 2) increased the sensitivity to 66.6%, leaving the specificity unchanged. A strong correlation was found between the SI of ER-D5 in E and F (p less than 0.001). The SI of ER-D5 in F sections was also well correlated with ER concentrations (p less than 0.001). These results suggest that immunocytochemical determination of ER-D5 in routine sections may be useful in retrospective studies of hormone dependence in breast cancer.

Frequency-dependent attenuation in breast tissue characterization.

The aim of this study is to establish whether an index derived from the slope of the frequency-dependent ultrasonic attenuation can provide quantitative information on normal and pathological breast tissue. Ultrasonic measurements were performed by using pulsed transmitted ultrasound in the frequency range 2-8 MHz. Thirty-three specimens were selected for their probable pathologic classification, by macroscopic observation, before ultrasonic study, and subsequently histologically classified. Ultrasonic results suggest the possibility that the examined specimens fall into four groups: (1) fat, fibroadenoma, giant fibroadenoma, infiltrating ductal carcinoma, medullary carcinoma; (2) infiltrating lobular carcinoma, tubular carcinoma, scirrhous carcinoma; (3) fibrosis; (4) fibrofatty tissue, fibrocystic disease. Correlative morphological studies indicate that the employed index can classify breast tissues on the basis of their cellular and fibrous composition and the inhomogeneity of their structure.

Orientation and frequency dependence of backscatter coefficient in normal and pathological breast tissues.

The backscatter coefficient was measured on five groups of normal and pathological breast specimens: (1) as a function of frequency (in the range 4-14 MHz) and (2) at a single fixed frequency (10 MHz), as a function of the angle of incidence between the beam and the specimen (approximately 60 degrees). The results of the study are discussed in relation to the content of cells and collagen fibers in breast tissues. The absolute value of backscatter coefficient is larger in tissues with a prevalence of collagenous fibers in comparison to tissues with only cells. In fibrofatty tissue, the inhomogeneity of the specimens is probably responsible for the highest backscatter value. The power law frequency dependence of the backscatter coefficient is of a diffractive nature in tissues characterized by only cells; in tissues with a strong prevalence of collagenous fibers, the power law frequency dependence increases. Periodicities in the angular patterns have been quantified by the autocorrelation functions for each group of specimens. The results of the study suggest a means for assessing tissue structure in normal and pathological breast tissue.

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.

Biological and immunological properties of the HMB-2 human melanoma cell line.

The HMB-2 human melanoma cell line derived from a lymph node metastatis is described. After long-term cultivation in vitro the cells retained their morphology, high growth rate, xenotransplantability into immunosuppressed mice and genotypic and phenotypic markers of human melanoma cells. Upon infection of the HMB-2 cells with temperature-sensitive mutant vesicular stomatitis virus (VSVts045) at nonpermissive temperature, a complemented virus pseudotype (VSV(HMB-2] was produced carrying assembled melanoma-associated antigens. Monoclonal antibodies prepared against HMB-2 cell membrane proteins and proteins of purified VSV(HMB-2) particles showed different reactivity with various human tumor cell lines and tissues: While the RG-12 anti-HMB-2 monoclonal antibody recognized a class II tumor-associated antigen present in melanoma and carcinoma tissues, the B-6 anti-VSV(HMB-2) antibody showed selective reactivity with melanoma cells.

Uneven distribution and significant concentration of apocrine metaplasia in lower breast quadrants.

Two hundred human breasts removed for clinical cancer by radical mastectomy were analyzed to determine whether apocrine metaplasia and apocrine cysts have predilective sites in the four mammary quadrants. One block of tissue randomly removed for each quadrant was examined in one or more histologic sections. The results showed a concentration of apocrine metaplasia and apocrine cysts in the lower mammary quadrants (17.5% versus 8.2%), which is highly significant (chi 2 = 15.25; P less than 0.001). The different distribution between breast cancer (showing predilection for the upper quadrants) and apocrine metaplasia is emphasized, together with the highly significant association between apocrine metaplasia and breast cancer. Based on these data, the usefulness of the clinical detection of cysts in the lower mammary quadrants and needle aspiration of their fluid for the morphologic or biochemical diagnosis of apocrine metaplasia in the screening and management of women at risk is suggested.

Characteristics of lymphomas in the BALB/cfRIII mouse strain.

The occurrence of 392 lymphomas in 1607 BALB/cfRIII mice from F0 to F45 is analyzed. Lymphoma incidence increased rapidly in the first 5 generations, reached a high plateau from F6 to F20 and then decreased slowly until it disappeared altogether. After F35, only sporadic cases of lymphoma have been observed. The morphologic characteristics of 350 of these lymphomas observed from F0 to F20 are described. Three main types of lymphomas have been recognized on the basis of gross morphology, histology and age: a) early, lymphocytic, with thymic involvement; b) early, lymphocytic, without thymic involvement; and c) late, histiocytic, without thymic involvement. The first 2 types are virus-induced and thymus-dependent, and the third type is both virus and thymus independent. Lymphomas without thymic involvement and histiocytic lymphomas increased with the generations. The source of causative virus, the interference with mammary tumors, and the possible cell types of origin of lymphomas are discussed.

Relationships between morphology and viral etiology in mammary tumors of BALB/cfC3H and BALB/cfRIII mice.

The morphologic pattern of mammary tumors induced by milk-transmitted C3H and RIII murine mammary tumor viruses (MuMTVs) in BALB/c hosts infected by foster nursing were investigated further an compared taking into account several tumor and host variables. Comparison of BALB/cfC3H and BALB/cfRIII mammary tumors under identical tumor and host conditions showed highly significant differences in subgross morphology and histologic structure. Differences in cytology were also observed. Data suggest that mammary tumor morphology in high cancer strain mice is controlled by the causative MuMTV.

Relationship of the degree and type of intraductal component with other morphologic findings in breast cancer.

The presence of mammary lobules around overt cancer and the amount and the type of intraductal tumor component (solid, comedo, cribrous, comedo-cribrous, papillary) were defined in 493 consecutive cases of infiltrating human breast cancer to analyze the association of these parameters with other morphologic parameters, such as productive fibrosis, lymphoid infiltrate and nuclear grade. Data showed a significant inverse relationship between degree of intraductal component and productive fibrosis (p less than 0.01) and direct relationships between a) presence of lobules and degree of intraductal component (p less than 0.02) and b) intraductal component of comedo type and lymphoid infiltrate (p less than 0.02). In addition, marked though not significant associations were observed between a) absence of lobules and intraductal component of papillary type (p less than 0.10) and b) nuclear grade 1 and intraductal component of the comedo type (p less than 0.10). Data suggest that cancers with a high degree of introductal component could have a lobular origin and papillary cancer could have a ductal origin. The other relationships observed might be useful in the subclassification of NOS (not otherwise specified) breast cancer.

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 less than 0.05), as well as with the presence of ductal and lobular proliferative changes in the collateral glandular tree (intraductal papillomas, P less than 0.01; atypical lobules, P less than 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.