Luciano Ozzello

The use of monoclonal antibodies to estrogen receptors (ER) for immunoperoxidase detection of ER in paraffin sections of human breast cancer tissue.

Two monoclonal antibodies against MCF-7 human estrogen receptors were used for immunoperoxidase staining of paraffin sections of human breast cancer tissue. The staining was predominantly located in the nucleus of epithelial cells. Variation in the staining intensity was observed among individual cells. A significant positive correlation between the number of positively stained cells and cytosol estrogen receptor content (fmol of bound estrogen/mg of protein) was observed. The potential and the limitations of the present techniques are discussed.

Lymphomas and pseudolymphomas of the alimentary tract: An Immunohistochemical study with clinicopathologic correlations

One hundred one lymphoproliferative lesions of the gastrointestinal tract (66 malignant lymphomas, 20 pseudolymphomas, and 5 borderline lesions) were reviewed. The pathologic features were compared to the clinical findings, with reference to differential diagnosis and prognosis. Special attention was paid to immunohistochemical features. The gross appearance was diagnostic in only a limited number of cases. Endoscopic biopsy alone was also of limited value because only a diagnosis of probability could be made in several cases. In most of them the definitive diagnosis had to be based on histologic examination of the resected specimen. Immunohistochemical examination was found to be very useful as an ancillary diagnostic technique. Malignant lymphomas displayed either cytoplasmic immunoglobulins with a monoclonal pattern (47.1 per cent) or a negative reaction, whereas the pseudolymphomas were generally characterized by polyclonal immunoglobulins. Ninety per cent of malignant lymphomas with cytoplasmic immunoglobulins contained lambda chains. The survival probability was found to be related to the size of the lesion, the depth of infiltration, and the immunohistochemical characteristics of the tumors. The histologic type of the lymphomas was of limited value as a predictor of prognosis in the present series.

Conjugation of interferon alpha to a humanized monoclonal antibody (HuBrE-3vl) enhances the selective localization and antitumor effects of interferon in breast cancer xenografts

Human mammary carcinoma xenografts (MCF-7) growing in nude mice were treated with natural interferon α (n-IFN-α) alone or conjugated to a humanized monoclonal antibody (MoAb) anti-breast mucin (HuBrE-3vl) or to irrelevant human IgG1κ. The IFN and the conjugates were administered as 20 intra-lesional (i.l.) injections to 1 of 2 xenografts in each mouse, or i.p. The growth inhibitory effects of HuBrE-3vl/nIFN-α were significantly greater than those of nIFN-α used as a single agent or conjugated to HuIgG1κ. These effects occurred locally in the tumors receiving i.l. injections and systemically, although to a slightly lesser extent, in the noninjected tumors of mice treated i.l. and in the xenografts of mice treated i.p. Biodistribution studies showed that the uptake of 125I-HuBrE-3vl/nIFN-α by the tumors 24 hours after i.l. or s.c. injection was greater than that of 125I-HuIgG1κ/nIFN-α,125 I-nIFN-α alone, or by normal tissues, documenting a tumor targeting effect and favorable tumor:normal tissues (T:NT) ratios. The targeting effects and the resulting tumor growth inhibition were favored by the IFN-mediated up-regulation of the HuBrE-3vl reactive antigen, which was more prominent after 3 weeks of treatment with HuBrE-3vl/nIFN-α. These results were superior to those we obtained previously with nIFN-α conjugated to another MoAb of the same group (Mc5). These studies point out the potential usefulness of HuBrE-3vl/nIFN-α for the local and systemic treatment of breast cancer lesions by providing a means of delivering high doses of IFN to the tumors while minimizing the amount of IFN binding to normal tissues.

Immunostaining of estrogen receptor in paraffin sections of breast carcinomas using monoclonal antibody D75P3 gamma: effects of fixation.

Immunostaining of estrogen receptor was carried out on paraffin sections of breast carcinomas using an anti-estrophilin monoclonal antibody (D75P3U03B3) and the avidinbiotin technique. The tumors were fixed in Bouins solution or in formalin for varying periods of time at room temperature or at 4°C. Best results were obtained following fixation in Bouins at room temperature or in formalin at 4°C. The staining was localized in the nuclei of carcinoma cells and was heterogeneous in intensity and extent. Prolonged fixation resulted in decreased immunoreactivity and in the appearance of nonspecific cytoplasmic and background staining. The estrogen receptor immunostaining on parafffin sections was found to be in concordance with that on frozen sections (Abbott ERICA) and with the steroid-binding assay (dextran-coated charcoal) in over 90% of the cases. This method is of easy and rapid execution and yields reliable and reproducible results

An immunohistochemical evaluation of progesterone receptor in frozen sections, paraffin sections, and cytologic imprints of breast carcinomas

Two monoclonal antibodies to progesterone receptor (PR), JZB39 and KD68, were used for the immunocytochemical visualization of PR in different kinds of breast cancer specimens including (1) cryostat sections of tumors frozen at −80°C; (2) paraffin sections of tumors fixed in formalin or in Bouins fixative for varying periods of time at room temperature or at 4°C; and (3) imprints and cryostat sections prepared from the tissue used for frozen section diagnosis and stored at −80°C after fixation in Zambonis solution. Sections of conventionally frozen specimens as well as imprints and cryostat sections stored for varying periods of time were stained with the peroxidase–antiperoxidase technique, whereas the avidin–biotin technique was used for paraffin sections. In all types of specimens the PR immunostaining was localized to the nuclei of carcinoma cells and displayed considerable heterogeneity both in intensity and in distribution of positive cells. Close correspondence was found between the different immunohistochemical techniques as well as between immunostaining and steroid‐binding assays. PR staining was more frequently positive in well‐differentiated than in moderately or poorly differentiated carcinomas, whereas no meaningful correlation was found between PR staining and extent of the disease. Similar results were obtained with the immunostaining of estrogen receptor in the same material using monoclonal antibodies H222 and D75P3γ. Thus, by choosing the technique that best suits the type of specimen available, it is possible to obtain valid information on the receptor status of any breast carcinoma, regardless of its size and clinical presentation.

Oestrogen receptors in human breast cancer

Histopathological factors which might explain inconsistency in published data attempting to correlate oestrogen receptor content (ER) and pathological features in primary breast tumours have been investigated in 194 cases. It was found, that unequal assessment of tumour type and of histological grading between observers is one important factor. In terms of grading, however, heterogeneity of growth pattern within the same tumour seems to be of greater significance. No significant correlation was found between histological type of tumour and ER content. However, a trend towards a correlation between the extent of tubule formation (as an indication of differentiation) and ER content was observed.

Immunostaining of Type IV Collagen and Smooth Muscle Actin as an Aid in the Diagnosis of Breast Lesions

▪ Abstract: The immunohistochemical staining patterns of type IV collagen (TIVC) and smooth muscle actin (SMA) were studied in benign and malignant breast lesions in order to assess their usefulness in the differential diagnosis of difficult lesions. Eighty‐six in situ breast carcinomas (8 with microinvasion; 1 diagnosed after immunostaining) and 58 invasive carcinomas, with associated benign lesions including 87 nonproliferative fibrocystic changes, 19 sclerosing adenosis, 17 atypical hyperplasias, 7 benign papillary proliferations, 6 radial scars, and 105 normal mammary tissue, were studied. TIVC and SMA were concomitantly positive throughout in 94% of benign tissues, in 16% of in situ carcinomas, but in none of the invasive carcinomas. Conversely, both markers were negative in 66% of invasive carcinomas, but in none of the benign breast tissue and none of the in situ carcinomas. In 6% of the benign tissues, in 21% of in situ carcinomas, and in 34% of invasive carcinomas only one of the markers was positive. In the remaining 63% of the in situ carcinomas there were discontinuities of the staining of both markers. It is therefore suggested that a diagnosis of invasive malignancy can be confirmed when both markers are negative, and ruled out when both markers are positive. This is particularly useful, in our experience, in the identification of small foci of invasion. The stains are useful when used in parallel, whereas they may be misleading when used singly. The staining pattern is not useful in the differential diagnosis of atypical hyperplasia and in situ carcinoma. ▪

Antiproliferative effects of natural interferon beta alone and in combination with natural interferon gamma on human breast carcinomas in nude mice

SummaryNude mice bearing bilateral xenografts of human breast carcinoma cells (MCF-7 and BT20) were treated with 2 or 4 5-day cycles of intralesional (i.l.) injections of human natural interferon beta (nIFN-β) alone or in combination with human natural interferon gamma (nIFN-γ). The injections were administered to only 1 of the 2 tumors in each animal, thus making it possible to assess at the same time local therapeutic effects in the injected tumors and systemic effects in the contralateral ones. When n-IFN-β was used as a single agent only mild local antitumor effects and virtually no systemic effects were observed. In contrast, the combined administration of nIFN-β/nIFN-γ produced marked antiproliferative effects, presumably as a result of the synergistic action of type I and type II IFNs. These effects ranged from complete regression documented histologically in 2 MCF-7 tumors to varying degrees of growth inhibition with persistence of residual microscopic or grossly detectable tumor. Local effects were more pronounced than systemic effects. The therapeutic efficacy of nIFN-β proved to be greater than that of recombinant interferon beta (rIFN-β). In MCF-7 tumors nIFN-β appeared to be less effective than nIFN-α, whereas the opposite was true for BT 20 tumors.

The use of natural interferon alpha conjugated to a monoclonal antibody anti mammary epithelial mucin (Mc5) for the treatment of human breast cancer xenografts

SummaryAn immunoconjugate composed of natural interferon α (nIFNα) bound in a noncleavable fashion to a monoclonal antibody (MoAb) recognizing a breast epithelial membrane mucin (Mc5) was used to treat xenografts of a human mammary carcinoma cell line (MCF-7) growing in nude mice. The immunoconjugate (nIFNα/Mc5) was administered as 20 intralesional (i.l.) injections to 1 of 2 xenografts in each animal. It was found that nIFNα/Mc5 produced a significant enhancement of the growth inhibitory actions of nIFNα on the injected tumors. Further enhancement was obtained when nIFNγ or nIFNγ together with Mc5 (at a dose 10 times larger than that present in nIFNα/Mc5) were added to the immunoconjugate. Biodistribution experiments showed that the uptake of125I-nIFNα/Mc5 by the tumors was greater and its elimination slower than for125I-nIFNα alone or conjugated to irrelevant mouse IgG1. In addition, the immunoconjugate up-regulated the antigenic expression of a breast epithelial membrane mucin by the carcinoma cells, an up-regulation which was not significantly different from that produced by nIFNα alone. The contralateral noninjected tumors exposed to systemic levels of the immunoconjugate showed an enhancement of antitumor effects, but to a lesser extent than the injected tumors. These findings suggest that the enhancement of the growth inhibitory action of the immunoconjugate was related to the specific binding of Mc5 which targeted the IFN to the carcinoma cells and impeded its elimination. It is likely that the targeting was favored by the IFN-mediated up-regulation of antigenic expression by the carcinoma cells, thereby producing a cascade of interrelated effects. The results of this study point out the feasibility and potential usefulness of IFN treatment by means of immunoconjugates as well as the worth of pursuing and improving this form of therapy.

Immunohistochemical assessment of estrogen and progesterone receptors in stored imprints and cryostat sections of breast carcinomas

A peroxidase-antiperoxidase technique was used to visualize estrogen and progesterone receptors in stored imprints and cryostat sections of breast carcinomas that were prepared at the time of biopsy or frozen section diagnosis. This was done to provide an alternate technique for the assessment of the receptor status of tumors that could not be adequately assayed with other biochemical or immunocytological methods. Fixation in Zambonis fixative followed by passage through cold methanol and acetone before storage at -80 C insured good preservation of the receptor proteins over extended periods of time (up to 56 weeks). Immunostaining of these stored preparations with monoclonal antibodies against estrogen receptor (H222) and progesterone receptor (JZB39 and KD68) showed a high degree of correspondence with immunocytochemical assays (ER-ICA and PR-ICA) and biochemical analysis. This technique is easy to perform and provides reliable information, even in tumors that are too small and/or ill defined to permit separate sampling for receptor assays.