Quantitation and localization of cycling tumor cells in pleomorphic adenomas and myoepitheliomas: an immunohistochemical analysis.

Previous investigations have suggested that in certain salivary gland tumors only nonluminal (myoepithelial-like) tumor cells proliferate and that this may have histogenetic implications; however, no quantitative assessment of the cycling component is available. We decided, therefore, to enumerate the cycling luminal and nonluminal cells in 15 pleomorphic adenomas and six myoepitheliomas by using an antibody to proliferating cell nuclear antigen. The mean percentages of cycling luminal and nonluminal cells in pleomorphic adenomas were 2.3 +/- 1.4 and 2.4 +/- 2.3, respectively, and 2.1 +/- 0.7 of the tumor cells in myoepitheliomas. Even though duct-like structures in pleomorphic adenomas are considerably separated by more numerous nonluminal cells and, therefore, cycling cells may seem fewer, both cell types proliferate at similar rates. The results indicate that nonluminal cells are not the sole proliferative component in salivary gland tumors.

Humoral immune response against p53 protein in patients with colorectal carcinoma.

p53 aberrations are frequent in colorectal carcinogenesis (40-70%). Because p53 gene mutations typically result in increased p53 protein concentration in tumor cells, this cellular protein might become immunogenic during tumor development. To test this hypothesis, serum p53 antibodies were quantitatively analyzed in 229 patients with colorectal cancer, using an immunofluorometric procedure. Circulating antibodies against p53 were found in 23% (53/229) of the patients. We quantified antibody concentrations in all positive sera and found that they varied from 300 to 500,000 arbitrary units/1. Sequential analysis of positive sera from 3 patients showed that p53 antibody concentrations change during the course of the disease, reflecting progression or regression. No association was found between the presence of p53 antibodies and age, sex, stage, histological grade and patient relapse-free or overall survival. These data demonstrate that antibody generation against the p53 tumor-suppressor protein is a relatively common event in colorectal cancer and that serological analysis for p53 antibodies may have some value for patient monitoring. The test has no value for prognosis.

Circulating antibodies against p53 protein in patients with ovarian carcinoma. Correlation with clinicopathologic features and survival.

BACKGROUND: Genetic alterations of the p53 tumor suppressor protein are the most frequent molecular events in human carcinogenesis. For as yet unknown reasons, mutant p53 often acts as an immunogen for autoantibody generation. These autoantibodies can be detected in the serum of cancer patients. The presence of such antibodies has been identified in a subset of patients with ovarian carcinoma, but their clinical significance has not been investigated. METHODS: Serum samples from patients with ovarian carcinoma were quantitatively analyzed for the presence of p53 autoantibodies with a time-resolved immunofluorometric procedure. Tumor p53 overexpression was assessed by immunohistochemical analysis of tissue sections. Kaplan-Meier survival curves were calculated for p53 antibody positive and negative patients, and the Cox model was used to evaluate the strength of the associations between the presence of serum p53 antibodies and cancer relapse or death, and also between the presence of such antibodies and other clinicopathologic features. RESULTS: p53 antibodies were detected in the serum of 41 of 174 patients with ovarian carcinoma (24%). Antibody levels ranged from a few hundred to 9 x 10(6) arbitrary Units/L, and fluctuated during the course of the disease. p53 antibody positive patients tended to have tumors overexpressing p53, but the association between the two parameters was not statistically significant (P = 0.13). There was also no association between the presence of p53 antibodies and clinical stage, tumor histologic type, or overall patient survival. However, these antibodies were more frequently present in patients older than 50 years (P = 0.001), in patients with moderately or poorly differentiated tumors (P = 0.001), and in patients who received chemotherapy (P = 0.015), and who suffered relapse after surgery (P = 0.018). In univariate analysis, p53 antibody positive patients were at an increased risk for relapse but not death. In multivariate analysis, the differences in disease free and overall survival between patients who were p53 antibody positive or negative were not statistically significant. CONCLUSIONS: p53 autoantibodies are found frequently in the serum of patients with ovarian carcinoma. The presence of such autoantibodies was associated with older patient age, more aggressive tumors, and reduced patient disease free survival. In multivariate analysis the prognostic value of p53 autoantibodies was not statistically significant.

Immunoelectron microscopy for chromogranin A in small cell neuroendocrine carcinoma of lung.

To see if immunoelectron microscopy can improve localization of neurosecretory granules, postembedding immunolabelling for chromogranin A was performed on 15 examples of small cell anaplastic (neuroendocrine) carcinomas primary in lung, five cases of bronchopulmonary carcinoids, and two cases of pheochromcytoma; both the carcinoids and pheochromocytomas had neurosecretory granule-rich cytoplasm by routine electron microscopy and some neurosecretory granules were identified in each of the small cell carcinomas. Immunolabeling for chromogranin A resulted in many colloidal gold particles over cytoplasmic secretory granules in both pheochromcytomas and four of the carcinoids. One carcinoid that was focally positive by immunoperoxidase staining was negative by immunoelectron microscopy. None of the 15 cases of small cell carcinoma stained for chromogranin A using immunoperoxidase techniques, but three had a small number of secretory granules weakly labeled by the anti-chromogranin A/colloidal gold complex. Immunoelectron microscopy, at least using standard glutaraldehyde fixation and epoxy resin embedding, does not increase the sensitivity of neurosecretory granule identification in small cell neuroendocrine carcinomas of the lung. Results of other studies of this neoplasm suggest that despite transcription of chromogranin genes, synthesis of the specific protein may occur to a limited extent or not at all.

Dynamics of follicular growth and atresia of large follicles during the ovarian cycle of the guinea pig: fate of the degenerating follicles, a quantitative study.

BACKGROUND: A quantitative integrated study of healthy ovarian follicles of different sizes and their mitotic activity and of clearly defined atretic stages of involuting large growing follicles at different stages of the guinea pig ovarian cycle is not available in the literature. We considered that such a study would reveal new aspects of ovarian tissue dynamics and provide new information in an organ with a continuous phenotypic transformation of its cellular components. METHODS: Ovaries from guinea pigs were removed on days 1 (opening of the vagina), 3, 6, 9, 13, and 16 of the cycle, and the following were measured in serial sections: (1) total number of healthy follicles falling into categories based on the volume occupied by granulosa cells, (2) total number of atretic follicles falling into clearly defined morphological stages of the degenerative and involutionary process affecting medium to large follicles, and (3) proportion of metaphase-arrested granulosa cells, after colcemid injection, in healthy follicles of different size categories. RESULTS: Dynamic patterns of follicular growth and degeneration were revealed that permitted the following main conclusions and observations: (1) small to middle-size follicles can reach the maximal category mass of granulosa mass within 6-7 days, and the number of granulosa cells can increase 6-7-fold during this interval, (2) the cohort that gives rise to 2-6 preovulatory follicles and to the large follicles that will undergo atresia during each cycle varied from 68 to 108 follicles, (3) cell death starts in the granulosa cell layers of large follicles even when neighbouring cells maintain a high mitotic activity and it spreads rapidly; dead granulosa cells are cleare by nucleolysis and cytolysis in the absence of blood leucocytes or neovascularization, (4) foci of atresia are observed also in a few preovulatory follicles, (5) antral cavities of follicles with dead granulosa cells in the process of being lysed shrink and are filled within 2-3 days with large fibroblast-like cells arising from phenotypic transformation of inner layers of theca interna, with no evidence of mitotic activity or angiogenesis; the outer layers of theca interna involute, and by progressive atrophy and a process of cell death, minute nodular structures arise with remnants of the ovum and zona pellucida, and (6) a transient wave of degeneration affects a proportion of small and middle-size follicles during the metestrous period. This process does not resemble the morphological phenomenology of follicular involution, which affects only large follicles. CONCLUSIONS: This study contributes to a fuller understanding of the dynamics and time relationships of follicular growth and loss in the guinea pig ovary and provides new morphogenetic information on the atretic process. It would be valuable for the design of experiments on endocrine and paracrine interactions involved in follicular growth and atresia.

Prediction of the outcome of transplantation in man by platelet adherence in donor liver allografts. Evidence of the importance of prepreservation injury.

We examined platelet adhesion in thirty human donor livers to determine if the degree of platelet adhesion could predict outcome of transplantation. Wedge liver biopsies were taken at the start of the donor operation (biopsy 1) and 1 hr after reperfusion in the recipient (biopsy 2). Biopsies were stained with a monoclonal antibody against platelet glycoprotein Ib and graded for platelet adhesion. Hematoxylin and eosin-stained sections were examined for polymorphonuclear leukocyte adhesion and necrosis. Platelet adhesion was much more frequent and extensive than expected in biopsy 1. Nine of 30 biopsies showed moderate or high-grade platelet adhesion. Thus in this study endothelial cell damage was present in about one-third of donors before the donor operation. The injury was not detectable by routine microscopic or clinical examination or biochemical tests. The degree of platelet adhesion in biopsy 1 predicted development of PMN adhesion and necrosis in biopsy 2 and postoperative transaminase concentrations and prothrombin times in recipients. During preservation and implantation some livers converted from low to either moderate or high grades of platelet adhesion. The grade of platelet adhesion in biopsy 2 predicted postoperative outcome as measured by transaminase and PT levels. Patients whose platelet grade converted to a higher level during preservation and implantation did not do as well as patients who remained at a low adhesion grade. These findings strongly suggest that the degree of platelet adhesion is an important determinant in assessing outcome and may provide a means of measuring the status of liver allografts prior to transplantation.

S-100 protein antibodies do not label normal salivary gland myoepithelium. Histogenetic implications for salivary gland tumors.

Neoplastically modified myoepithelial cells have a key role in developing the histologic characteristics of some salivary gland tumors. S-100 protein expressed in certain of these tumors is suggested to support this role, as the principal component in the human salivary gland reported to be S-100 protein-positive is myoepithelium. Confirmation of such an important aspect is required. Immunoperoxidase staining of parotid salivary gland shows considerably different patterns obtained with antibodies to S-100 protein, neuron-specific enolase, and neurofilaments compared with those for muscle-specific actin and cytokeratin 14; many more cells and their processes associated with acini and ducts are evident with the latter two antibodies. Double immunofluorescent staining with antibodies to either S-100 protein or neuron-specific enolase combined with muscle-specific actin does not reveal colocalization of these antigens in myoepithelial cells. The former localize only to nerve fibers adjacent to, but separate from, acini, and the latter only to myoepithelial cells. It is apparent that S-100 protein staining of the rich network of unmyelinated nerves in the interstitial tissues, evident ultrastructurally, has been misinterpreted as myoepithelium. This result has important implications for histogenetic classifications of salivary gland tumors.

Immunohistochemical staining of germ cell tumors and intratubular malignant germ cells of the testis using antibody to placental alkaline phosphatase and a monoclonal anti-seminoma antibody.

Antibody to placental alkaline phosphatase (PLAP) has been previously used in immunoperoxidase (IMP) staining studies of germ cell tumors and intratubular malignant germ cells (ITMGC) of the testis, the latter believed to be the precursor of these tumors. In this study, we compared staining by IMP using monoclonal antibody (mAb) and polyclonal antibody to PLAP with that seen using a mAb, M2A, which was previously shown to react with testicular seminomas and ITMGC. Antibody to PLAP and M2A reacted with different cellular components, as assessed by IMP staining of placenta and prepubertal testis and by Western blotting of seminoma lysates. Antibody to PLAP stained pure seminomas (seven of seven), pure embryonal carcinomas (four of four), and the seminoma (three of three) and embryonal carcinoma (six of six) components of mixed testicular germ cell tumors. M2A stained pure seminomas (26 of 26) and the seminoma component (three of three) of the mixed tumors, but failed to stain pure embryonal carcinomas (zero of four) or the embryonal carcinoma component (zero of five) of the mixed tumors. Both antibody to PLAP and M2A stained ITMGC of the testis. Since M2A stained seminomas and ITMGC but not embryonal carcinomas, seminomas would appear to be more closely related to ITMGC than embryonal carcinomas. This result has led us to speculate on the histogenesis of testicular germ cell tumors.

The mechanism of ceruminolysis.

In a previous study comparing the efficacy of a selection of commonly used ceruminolytics, the authors noted that aqueous-based preparations, and in particular solutions of sodium bicarbonate, were more effective in disintegrating cerumen than most organic-based preparations. In that study, the authors also observed that not only had the wax truly disintegrated following exposure to the aqueous-based preparations, but also that a marked degree of swelling of the wax spheres had occurred with these preparations. In this paper the mechanism of ceruminolysis was investigated by means of a number of commonly available histological techniques. Our findings show that desquamated sheets of corneocytes are the major constituent of cerumen plugs and provide the structural framework of the wax bolus. Ceruminolytics work by hydrating the keratin cells of these sheets of desquamated stratum corneum and subsequently inducing keratolysis, with disintegration of the wax.

A novel maturation marker for human Sertoli cells.

The maturation of Sertoli cells at puberty is critical for the initiation and maintenance of spermatogenesis. Little is known about the factors which control Sertoli cell maturation. We have examined Sertoli cells in testicular specimens from 44 subjects, ranging in age from 6 months to 67 years, for reactivity with a mouse monoclonal antibody (M2A) using the immunoperoxidase reaction. We found positive reactivity with prepubertal but not postpubertal Sertoli cells, suggesting that the antigen defined by M2A was a marker of immature Sertoli cells. This marker may be useful for studying the factors which influence Sertoli cell maturation during development of the testis.

Nuclear antigens in neoplastic lymphocytes of B cell and T cell non-Hodgkin's lymphomas.

Gross nuclear morphology is a major diagnostic feature in the identification of subtypes of non-Hodgkin's lymphoma (NHL). The authors have shown that the size, shape, and chromatin distribution of the lymphocyte nuclei vary extensively both within and between samples of a subtype, and have proposed that the variations may reflect qualitative and quantitative differences in extrachromatinic components. To test this hypothesis, the organization of individual nuclear antigens in NHL and in reactive hyperplasia biopsies was examined by immunofluorescence labeling of frozen sections with previously characterized monoclonal antibodies. The results have been correlated with observations of the staining patterns produced by the antibodies in mitogenically stimulated human peripheral blood lymphocytes. Labeling pattern and intensity with each antibody were consistent between preparations of blood lymphocytes, and all four antibodies labeled all blood lymphocyte samples tested. In contrast, only 15% of the 53 biopsies were labeled by all four antibodies, although all were stained by anti-peripherin, nearly 80% by I1, and almost 60% by PI1. Antibody PI2 labeling was detected in only 20% of the samples. Variation in labeling intensity was equally extensive both within and between biopsy samples. In general, there was little homogeneity between samples of an NHL subtype as to which antigens were detected, their labeling intensity, or their pattern of intranuclear distribution. These observations are consistent with earlier reports of significant diversity in the morphology of nonchromatin components in such samples. The data support the proposition that the heterogeneity of gross nuclear morphology in nuclei of NHL biopsies may be due in part to disordered expression or abnormal organization of nuclear proteins.

Corneocyte architecture in the human external auditory canal.

Using skin dissected from the external auditory canals of fresh cadaveric temporal bones, the structure of the stratum corneum was investigated by the techniques of alkaline expansion of cryostat sections, silver staining of epidermal sheets, and scanning electron microscopy. Ordered epidermal column formation was observed in the stratum corneum from skin obtained from both the bony and cartilaginous portions of the canal. Occasionally, in areas of skin where the stratum corneum was considerably thickened, this vertical organization was lost, and the corneocytes randomly arranged. This orderly cellular architectural arrangement is a feature of the non-migratory skin of lower mammals, and has also been reported to occur in some areas of human skin. The functional relevance of this finding in the skin of the external canal is that the whole of the stratum corneum must migrate as one in order to preserve a regular vertical structure, suggesting that epidermal migration occurs in the deeper layers of the meatal epidermis.

Production of a monoclonal antibody specific for seminomas and dysgerminomas.

A monoclonal antibody (M2A, IgG2a) was produced against a cultured human ovarian epithelial adenocarcinoma cell line, HEY. Monoclonal antibody M2A reacted with a glycoprotein of molecular weight 40,000 on the surface of HEY cells. The affinity constant of the monoclonal antibody M2A for HEY cells was 10(9) M-1, and the number of binding sites on HEY cells was 2 X 10(4) per cell. The monoclonal antibody produced positive immunoperoxidase staining of fetal (but not adult) testis and of seminomas and dysgerminomas but did not stain various normal adult tissues or other gonadal or extragonadal tumors. Monoclonal antibody M2A may be useful for confirming a histological diagnosis of seminoma and dysgerminoma.