M. Vierbuchen

Comparative histological, histochemical, immunohistochemical and biochemical studies on oestrogen receptors, lectin receptors, and Barr bodies in human breast cancer.

The present study performed on a total of 567 cases of human female breast cancer compares the results of the biochemical assay (dextran-coated charcoal assay=DCC) for oestrogen receptor (ER) with those of several morphological methods developed for the detection of the ER or for the prediction of prognosis by use of other systems (FSA= fluorescent ligand binding assay, ER-ICA=monoclonal antibody assay for ER, LRA=lectin receptor assay using peanut agglutinin, and Barr body estimation). Whereas no correlation at all was observed among the results of the DCC and those of the FSA and Barr body estimation, the ER-ICA and the LRA showed an unanimous tendency towards higher values of ER with increasing intensity of the staining product. The results of the ER-ICA may be expressed by an immuno-reactive score (IRS) calculated from the staining intensity (SI) and the percentage of positive cells (PP). The morphological methods are evaluated with special regard to their correlation with the DCC, their theoretical basis, and their practical application. In summary, the ER-ICA appears to be the sole method directly visualizing the ER protein and - in contrast to the DCC - is therefore completely independent of the content of endogenous or exogenous oestrogens in the tumor tissue. The LRA provides valuable additional information concerning tumour differentiation

Detection of squamous cell carcinoma antigen in normal squamous epithelia and in squamous cell carcinomas of the uterine cervix.

Squamous cell carcinoma (SCC) antigen is a subfraction of tumor antigen TA‐4 isolated from a cervical squamous cell carcinoma. The specificity of SCC antigen and the factors influencing its release into serum were evaluated. Antigen concentrations were measured in 157 tissue extracts and in 188 sera of patients with nonmalignant or malignant gynecologic diseases. A commercial radioimmunoassay based on polyclonal antibodies (Abbott Laboratories, North Chicago) was used. Cytosol concentrations were significantly higher (P < 0.005) in normal squamous epithelia (x̃ = 6040 ng/mg cell protein [CP]) and in squamous cell carcinomas (x̃ = 2483 ng/mg CP) of the exocervix than those in normal columnar epithelia and in adenocarcinomas of the endocervix, endometrium, ovary, and breast (x̃ = 1–508 ng/mg CP). Despite the high antigen concentrations in normal squamous epithelia, elevated serum levels (>2.5 ng/ml) were almost exclusively found in patients with cervical squamous cell carcinomas. The sensitivity of SCC antigen as a marker for primary carcinomas was 61%, increasing from 29% in Stage I to 89% in Stage IV. The positivity rate was higher in women with well‐differentiated (78%) and moderately differentiated carcinomas (67%) than in those with poorly differentiated tumors (38%). The results show that SCC antigen is not tumor specific. The release into serum is independent of local tissue content, but is apparently influenced by the infiltrative growth, the mass, and the degree of histologic differentiation of the tumor.

Immunohistochemical study of distribution of estrogen receptors in corpus and cervix uteri

SummaryAn immunohistochemical assay based on monoclonal antiestrophilin antibodies has been used to localize estrogen receptor (ER) in frozen sections of normal human endometrial, myometrial and cervical tissues from menstruating, hormonally treated, pregnant and postmenopausal women. Specific staining was confined to the cellular nuclei. In proliferative phase endometrium, postmenopausal emdometrium, and endometrium from patients treated with hormone ERs were easily detected in most glandular and stromal cells. After ovulation and in early pregnancy a quick and distinct decrease of ER expression was noted. This was especially the case with the more superficial layers of endometrium (endometrium functionals), the majority of whose cells had either weak localization of ER or none at all. In the endometrium basalis, however, the reduction of ER localization turned out to be more moderate. More then half of the epithelial and stromal cells displayed nuclear staining, partly strong. The myometrium of the corpus uteri showed a similar ER localization and dependence on hormonal stage when compared with the endometrium functionalis. The endocervical mucosa displayed a high degree of ER expression in the proliferative phase in postmenopausal women and in women who had been treated with hormones. Unlike the endometrium and myometrium, the endocervical glands underwent minimal changes in nuclear ER content during the menstrual cycle. Although the endocervical stroma showed cyclic alterations in ER levels, their reduction after ovulation was less marked than in the corresponding endometria. In cervical squamous epithelium ER localization was predominantly confined to the basal layers. In the course of cellular maturation, specific nuclear staining vanished. In the proliferative phase, after the menopause and in early pregnancy, the basal, parabasal and intermediate cells were specifically stained. In the postovulatory phase. However, nuclear staining was confined to the basal and parabasal cells. Hormonally treated squamous epithelia almost completely lacked nuclear ER localization.

Differentiation of plasma cell infiltrates in the bone marrow. A clinicopathological study on 80 patients including immunohistochemistry and morphometry.

In 80 patients immunohistochemical, morphometrical and clinical studies were performed on routinely referred trephine biopsies of the bone marrow showing an abnormal increase in plasma cells. From the approximately determined density of plasma cell infiltrates two main groups were distinguished, the first with an involvement exceeding 20% and the second with less than 10% of the total marrow area involved. The first group (n=30; 324±130 plasma cells per square millimeter bone marrow) consisted of patients with frank malignant myeloma (MM) by clinical and histomorphological diagnosis. The second group (n=50; 132±54 plasma cells per square millimeter bone marrow) with plasmacytic differentiation of infiltrates, had to be further divided into one component with evidence for initial or residual MM following chemotherapy (n=27), another with obviously monoclonal gammopathy of undetermined significance - benign monoclonal gammopathy (BMG,n=6), and a final set of cases with a reactive plasmacytosis mostly associated with an inflammatory condition (n=17). There was an excellent agreement between the intracellular immunoglobulin staining as defined by the immunoperoxidase technique and the serum or urinary M-component detected by immunoelectrophoresis. In MM significant correlations were found between osteoclastic activity (number of osteoclasts specifically stained by acid phosphatase) per trabecular bone area, presence of lytic bone defects and the density of plasma cell infiltrates in the marrow. This latter feature corresponded well with the titer of secreted serum M-components measured by quantitative immunoelectrophoresis. Using morphological data alone, BMG cases could not be discriminated with any certainty from initial or residual plasmacytic MM. They consequently need a prolonged clinical follow up to clarify the nature of the lesions.

Immunohistochemical detection of progesterone receptor in formalin-fixed and paraffin-embedded breast cancer tissue using a monoclonal antibody.

SummaryThe potential for immunohistochemical detection of progesterone receptors (PRs) in routinely formalin-fixed and paraffin-embedded cancer tissues by use of the monoclonal antibody Mi 60-10 (mPR1, Dianova GmbH, Hamburg) was evaluated. The PR content of breast cancer tissue was investigated in 170 cases. A positive reaction to Mi 60-10 was found exclusively in the nuclei of benign or malignant epithelial cells. The distribution of PRs was heterogeneous. Immunohistochemical reaction was scored by multiplying the percentage of positive tumour cells by their prevalent degree of staining (Immunoreactive Score or IRS). The IRS values of formalin-fixed tissues (n=170) were compared with those in snap frozen tissues (n=82), with the PR content assayed by a DCC (dextran-coated charcoal) method (n=170), with histopathological grading according to Bloom and Richardson and with the menopausal status of the patient. There was an acceptable ranked correlation (r=0.74) between IRS in formalin-fixed and paraffin-embedded parts and snap frozen parts of the same carcinoma. A good correlation (r=0.72) was also found, when the semiquantitative results of immunohistochemical PR detection in formalin-fixed and paraffin-embedded tissues were compared to PR concentrations measured by a DCC method in tumor cytosols. There was an 80% concordance between the two methods for qualitative discrimination of PR-negative and PR-positive carcinomas. IRS correlated significantly with the degree of histological differentiation of the tumors (P<0.001) but not with the menopausal status of the women (P>0.05). Storage of paraffin-embedded tissues did not impair PR detection, for up to at least 5 years. Fixation of tissues in formalin only decreased the immunohistochemical detection rate if fixative acted for more than 24 h.

Die Expression des epidermal growth factor (EGF), seines Rezeptors (EGF-R) und des Onkogens c-erb B 2 im primären Mammakarzinom

Zahlreiche autokrine Wachstumsfaktoren und ihre Rezeptoren weisen grose Sequenzhomologien zu Onkogenen auf, denen eine wichtige Rolle bei der malignen Transformation von Zellen zugesprochen wird. Die Wirkung des epidermal growth factor (EGF), einem potenten Mitogens fur das Brustdrusengewebe, wird uber seinen Rezeptor (EGF-R) vermittelt. Die Expression des EGF-R und des vom strukturahnlichen c-erb B2 kodierten Proteins (p185) scheint im Mammakarzinom vermehrt. Dies soll Ausdruck einer ungunstigen Prognose sein.

Immunhistochemische und biochemische Steroidrezeptoranalyse in Mammakarzinomen

Immunhistochemische Untersuchungen uber das Vorkommen des Ostrogen- (ER) und Progesteron-Rezeptors (PR) wurden an 70 Mammakarzinomen durchgefuhrt und die Resultate mit der biochemischen Rezeptoranalyse (DCC-Verfahren) korreliert. Der immunhistochemische Nachweis des Ostrogenrezeptorproteins erfolgte am Kryostat-, die Darstellung des Progesteronrezeptorproteins am Kryostat- und Paraffinschnitt mit Hilfe monoklonaler Antikorper (ER: ER-ICA, Abbott GmbH, PR: mPR1, Dianova GmbH). Die immunhistochemische Farbung wurde semiquantitativ bewertet durch Ermittlung eines immunreaktiven Scores (IRS), welcher den Prozentsatz spezifisch gefarbter Zellen und die Farbeintensitat berucksichtigt und Werte zwischen 0 und 12 vergibt. Als rezeptornegativ wurden Tumoren mit einem IRS von 0 oder 1 bzw. mit einer Rezeptorkonzentration von weniger als 20 fmol/mg Zellprotein eingestuft.

Immunhistochemische Lokalisation des Östrogenrezeptors am Uterus

Ffille ohne gr6f3ere Anteile von Nichtkarzinom mit ER-DCC zuverlfissig auszuwerten, da Stroma und Myometrium auch bei rezeptornegativem Karzinom positiv reagieren; diese Differenzierung gelingt jedoch nur mit dem ER-ICA-Verfahren. Somit liefert der immunzytochemische Nachweis des Ostrogenrezeptors mit hoher Zuverlfissigkeit dieselben Aussagen wie die Zytosolanalyse beim Mammakarzinom, erm6glicht eine genauere Beurteilung beim Endometriumkarzinom. ER-ICA kann auch bei so kleinen Gewebeproben durchgefiihrt werden, dab sie fiir ER-DCC nicht ausreichen. Transport und Aufbewahrung sind ftir beide Verfahren gleich (Notwendigkeit einer Ktihlkette, Einfrieren in fltissigem Stickstoff und Aufbewahren bei -80 ~ Laboraufwand und Zeitbedarf sind ftir ER-ICA geringer.

Histochemische Untersuchungen über die hormonelle Steuerung von Lektinrezeptoren im Brustdrüsengewebe der Ratte

(K61n): Histochemische Untersuchungen fiber die hormonelle Steuerung von Lektinrezeptoren im Brustdriisengewebe der Ratte Im normalen und karzinomatfs verfinderten Brustdriisengewebe finden sich Glykosubstanzen, die das Disaccharid fl-D-Gal(1-3)GalNAc in freier und sialinsfiuresubstituierter Form enthalten. Histochemisch wurden daher diese Kohlehydratsequenzen mit FITCbzw. Rhodamin-markiertem Erdnuglektin (peanut agglutinin, PNA), das eine hohe Affinit~it zu dem Disaccharid aufweist, direkt (freie Rezeptoren) oder nach Neuraminidasebehandlung (neuraminsfiuresubstituierte Rezeptoren) am formalinfixierten Brustdrfisengewebe der Ratte dargestellt, mit dem Ziel ihre Hormonabh~ngigikeit zu prfifen. Nach Ovarektomie kam es im Brustdrfisengewebe geschlechtsreifer Ratten zu einer Verminderung der PNA-Bindungsstellen. Wurden nun ovarektomierte Tiere mit 17/%Ostradiol behandelt, so fand sich neben einer Proliferation des Brustdrfisenparenchyms auch eine vermehrte Biosynthese yon PNA-Bindungsstellen, die im apikalen Bereich der Epithelzellen und im Sekret lokalisiert waren. Im Gegensatz zum 17/3-13stradiol ffihrte die alleinige Gabe yon Progesteron am ovarektomierten Tier zu keiner Stimulation der PNA-Rezeptorbiosynthese. Die durch 17/3-Ostradiol induzierte Expression yon PNA-Bindungsstellen konnte durch die gleichzeitige Gabe des 13strogenantagonisten Tamoxifen fast vollstfindig unterdriickt werden. Dies war yon einer Hemmung der Proliferation des Brustdrfisenepithels begleitet. Die durch 17/3-Ostradiol stimulierte Biosynthese des PNA-Rezeptors, wurde weiterhin durch Actinomycin D und Cycloheximid gehemmt, so dab angenommen werden kann, dab die hormoninduzierte Bildung yon PNA-Bindungsstellen fiber eine Stimulation der RNAund Proteinsynthese verlfiuft. In diesen Stoffwechsel greift offenbar auch das Prolactin ein, da nach Hemmung der Prolactinsekretion mit Bromocriptin eine Reduktion der PNA-Rezeptorbiosynthese nach Ostrogenstimulation beobachtet wurde. Allerdings vermochte die alleinige Gabe yon Prolactin am ovarektomierten Tier nicht die Bildung von PNA-Bindungsstellen zu induzieren. Erst die simultane Verabreichung von Prolactin und 17/%Ostradiol unter Ausschaltung der endogenen Prolactinsekretion mit Bromocriptin resultierte wieder in einer gesteigerten Biosynthese von PNA-Bindungsstellen. Zusammenfassend ergaben die Tierexperimente, dab die Expression yon PNA-Bindungsstellen im Brustdrfisengewebe eng an sekretorische und z. T. proliferative Vorgfinge gekoppelt ist. Weiterhin zeigten die Untersuchungen, dal3 im Brustdr~sengewebe die PNA-Bindungsstellen das Endprodukt eines hormonmodulierten Stbffwechselweges darstellen, ffir dessen Steuerung dem Ostrogen im Zusammenspiel mit dem Prolactin eine Schlfisselstellung zukommt.