WNT pathway and mammary carcinogenesis: loss of expression of candidate tumor suppressor gene SFRP1 in most invasive carcinomas except of the medullary type.

Secreted Frizzled-related protein 1 (SFRP1) encodes a member of a protein family that contains a cysteine-rich domain similar to the WNT-binding site of Frizzled receptors and regulates the WNT pathway. The WNT pathway is frequently altered in human cancers. We have defined the pattern of SFRP1 mRNA expression in the progression of breast cancer. We show that SFRP1 is expressed in the epithelial component of normal breast, in the in situ component of ductal carcinomas and is lost in more than 80% of invasive breast carcinomas except the medullary type. Loss of SFRP1 expression is correlated with the presence of hormonal receptors. Conversely, the maintenance of SFRP1 in carcinomas is correlated with the presence of lymphoplasmocytic stroma. No significant association was observed between SFRP1 status and the level of apoptosis in tumoral cells.

Bilateral laparoscopic adrenalectomy for congenital adrenal hyperplasia with severe hypertension, resulting from two novel mutations in splice donor sites of CYP11B1.

We present an in vivo and in vitro study of congenital adrenal hyperplasia in a patient with 11beta-hydroxylase deficiency. Sequencing of the CYP11B1 gene showed two new base substitutions, a conservative 954 G-->C transversion at the last base of exon 5 (T318T), and a IVS8 + 4A-->G transition in intron 8. In addition, two polymorphisms were found in exons 1 and 2. The genetically female patient was raised as a male because of severe pseudohermaphroditism. Glucocorticoid-suppressive treatment encountered difficulties in equilibration and compliance, resulting in uncontrolled hypertension with pronounced hypertrophic cardiomyopathy. At 42 yr of age the occurrence of central retinal vein occlusion with permanent loss of left eye vision led to the decision to perform bilateral laparoscopic adrenalectomy. Surgery was followed by normalization of blood pressure and good compliance with glucocorticoid and androgen substitutive therapies. In vitro, adrenal cells in culture and isolated mitochondria showed extremely low 11beta-hydroxylase activity. Analysis of adrenal CYP11B1 messenger ribonucleic acid (mRNA) by RT-PCR and sequencing showed the expression of a shorter mRNA that lacked exon 8 and did not contain either the exon 5 mutation or the exon 1 and 2 polymorphisms. This suggested that one CYP11B1 allele carried the intron 8 mutation, responsible for skipping exon 8. The other allele carried the exon 5 mutation, and its mRNA was not detectable. Western blot analysis showed weak expression of a shorter CYP11B immunoreactive band of 43 kDa, consistent with truncation of exon 8. Thus, bilateral adrenalectomy in this patient allowed effective treatment of severe hypertension and helped in understanding the mechanisms and physiopathological consequences of two novel mutations of CYP11B1.

A large multiple endocrine neoplasia type 1 family with clinical expression suggestive of anticipation.

We describe a large multigenerational multiple endocrine neoplasia Type 1 (MEN1) family with clinical expression suggestive of anticipation. In the second and third generations, two deceased obligate gene carriers died at the ages of 85 and 76 without the history of MEN1, whereas two other living gene carriers above the age of 65 have had no clinical evidence of MEN1 to date. In the fourth generation, eight members were affected, with four having severe MEN1-related and atypical malignancies: a case of metastatic endocrine pancreatic tumor, two cases of metastatic thymic carcinoids, and a case of spinal ependymoma. In the fifth generation, all five patients were below the age of 22 when the disease was detected. MEN1 was confirmed in the family by linkage analysis using MEN1-linked microsatellite markers and by identification of a nonsense mutation in the MEN1/menin gene. Alleotyping showed loss of heterozygosity (LOH) involving the wild-type alleles in seven tumors in the family including the ependymoma, which is the first MEN1-related case that shows genetic abnormality in chromosome 11q13, suggesting that MEN1 gene might be involved in the tumorigenesis of a subset of ependymomas. In relation to clinical anticipation, repeated expansion studies were carried out but failed to detect any expansion. We conclude that this is a unique MEN1 family and that an unknown genetic mechanism might be contributing to the anticipation phenomenon. We demonstrate in this family that all gene carriers, including the very young members, will need close and careful follow-up.

Cushing's syndrome due to a gastric inhibitory polypeptide-dependent adrenal adenoma: insights into hormonal control of adrenocortical tumorigenesis.

We studied a patient with food-induced, ACTH-independent, Cushing's syndrome and a unilateral adrenocortical adenoma. In vivo cortisol secretion was stimulated by mixed, glucidic, lipidic, or proteic meals. Plasma ACTH levels were undetectable, but iv injection of ACTH stimulated cortisol secretion. Unilateral adrenalectomy was followed by hypocortisolism with loss of steroidogenic responses to both food and ACTH. In vitro, cortisol secretion by isolated tumor cells was stimulated by the gut hormone gastric inhibitory polypeptide (GIP) and ACTH, but not by another gut hormone, glucagon-like peptide-1 (GLP-1). Both peptides stimulated the production of cAMP but not of inositol 1,4,5-trisphosphate. In quiescent cells, GIP and ACTH stimulated [3H]thymidine incorporation and p42-p44 mitogen-activated protein kinase activity. GIP receptor messenger ribonucleic acid (RNA), assessed by RT-PCR, was highly expressed in the tumor, whereas it was undetectable in the adjacent hypotrophic adrenal tissue, in two adrenal tumors responsible for food-independent Cushing's syndrome, and in two hyperplastic adrenals associated with ACTH hypersecretion. In situ hybridization demonstrated that expression of GIP receptor RNA was confined to the adrenocortical tumor cells. Low levels of ACTH receptor messenger RNA were also detectable in the tumor. We conclude that abnormal expression of the GIP receptor allows adrenocortical cells to respond to food intake with an increase in cAMP that may participate in the stimulation of both cortisol secretion and proliferation of the tumor cells.

Enhanced chemiluminescence: a high-sensitivity detection system for in situ hybridization and immunohistochemistry.

The breakthrough of chemiluminescence in the field of solution immunoassays and transfer membranes prompted us to explore whether a light-based detection system could provide a gain in sensitivity over chromogenic and FITC markers for nucleic acid and protein detection on histological preparations. A Hamamatsu device and an enhanced chemiluminescence (ECL) luminol substrate of the peroxidase were used to detect epithelial and endothelial components by immunohistochemistry (IHC) and for in situ hybridization (ISH) of papilloma virus DNA. The accuracy of the signal was compared to that obtained with DAB-peroxidase, silver-enhanced DAB-peroxidase, NBT-BCIP-alkaline phosphatase, and FITC. Our results demonstrated the feasibility and high sensitivity of luminescence detection for histological preparations. In part due to the ultrasensitive videocamera and photon-counting imaging, interpretable and reproducible results were obtained within counting times shorter than 5 min, and with dilutions of the primary antibodies 100- to 10,000-fold greater than those used for chromogenic and FITC reactions. As for ISH, with identical concentrations of the HPV 18 DNA probe on HeLa cells, labeling was apparent by luminescence but undetectable with the chromogen. The morphological resolution allowed a discriminatory analysis of the signal. Therefore, at the light microscopic level, enhanced chemiluminescence offers an appealing alternative to FITC and chromogenic markers for detection and quantification of low-concentration molecules.

F(ab) secondary antibodies: a general method for double immunolabeling with primary antisera from the same species. Efficiency control by chemiluminescence.

We propose a general solution to the problem of using antibodies originating in the same species for double immunohistochemical labeling. It relies on the use of a two-step protocol in which a secondary polyclonal monovalent F(ab) antibody present in the first step blocks access in the second of the secondary antibody to the primary antibody, which is continuously present from the first step. The monovalence of the F(ab) fragment eliminates the possibility of its linking the primary antibody from the second step. We designed two efficiency tests to explore the limits of the method by the very sensitive chemiluminescent system applied to sections of human pituitary tissue. They confirmed both the validity of the method and the necessity of adapting working conditions to obtain a complete lack of interference.

TUNEL apoptotic cell detection in tissue sections: critical evaluation and improvement.

TUNEL, i.e., terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling, has become a widely used staining method to assist in detection of apoptotic cells in tissue sections. However, despite its apparent simplicity, this technique has led to considerable disappointment because of its serious limitations in sensitivity and, even more, in specificity. We reviewed the limitations and artifacts of TUNEL and designed a comprehensive protocol to reassess the various procedures in use for five crosslinking and/or precipitating fixatives. By introducing microwave heating in extreme pH-value solutions (pH 3 for formalin and pH 10.6 for Bouin's fixative) coupled with proteolysis, we obtained an intense staining of 70-80% of apoptotic cells and bodies on archival tissue blocks, with little or no background. Owing to the enhanced sensitivity, early stages of apoptosis could be visualized and may enlarge our vision of the apoptotic cell beyond the mere image of shrinkage necrosis. We conclude that TUNEL remains a technique as useful as it is delicate, requiring critical interpretation of the staining. This study points out that, on archival tissues, despite the technical improvements we propose no protocol can be the final answer to all problems. Technique must be readjusted for any variation in tissue processing. However, step-by-step progress has rendered this method not only applicable but also performable within the constraints of archival surgical pathology specimens.

In situ apoptotic cell labeling by the TUNEL method: improvement and evaluation on cell preparations.

TUNEL (terminal deoxynucleotidyl transferase-mediated dUTP nick end-labeling) is a method of choice for rapid identification and quantification of the apoptotic cell fraction in cultured cell preparations. However, TUNEL application has been restricted to a narrow spectrum of sample conditions, and only detergents have been proposed as labeling enhancers. This study was aimed at extending TUNEL to variously fixed cells and improving TUNEL sensitivity by optimized pretreatments, the specificity being assessed by reference to the apoptotic morphology. Comparative TUNEL was performed with three protocols on CEM-C7 cells, a model of glucocorticoid-induced apoptosis. Samples were submitted to six modalities of fixation and TUNEL was performed after each of the following conditions: no pretreatment; detergent permeabilization; proteolytic digestion; microwave irradiation; and a recently published combination of the latter two. The proportion of TUNEL-stained elements within the cell fraction, with and without apoptotic morphology, was quantified. Our results showed that: (a) with an adequate pretreatment, reliable TUNEL can be obtained after each fixative tested; (b) detergent was inefficient in improving sensitivity; (c) whatever the fixation, microwave pretreatment provided the best TUNEL sensitivity without notable loss of specificity; (d) under adaptive technical conditions, TUNEL can be associated with detection of various proteins by double labeling; and (e) the existence of a limited population of intensely TUNEL-positive cells that lacked apoptotic morphology contributes to the current debate about a preapoptotic state.

Comparison of 35S and chemiluminescence for HPV in situ hybridization in carcinoma cell lines and on human cervical intraepithelial neoplasia.

For in situ hybridization (ISH), development of sensitive, nontoxic alternatives to the use of radioactivity is a constant concern. In this trend, and close to chromogenes and fluorophores, chemiluminescence appears an attractive method. A first positive experience in immunocytochemistry and in ISH, by using the enhanced luminol as luminogene substrate for horseradish peroxidase (HRP) led us to compare the sensitivity of 35S autoradiography and chemiluminescence. For this purpose, we used three human carcinoma cell lines, CaSki [400-600 copies of human papilloma virus (HPV) 16], HeLa (10-50 copies of HPV 18), and SiHa (1-5 copies of HPV 16), and 40 biopsy specimens of human cervical preneoplastic and neoplastic lesions. We performed ISH by using HPV cDNA biotin-labeled probes, detected by a two-step immunocytochemical reaction, the secondary antibodies being either 35S-labeled for autoradiography or HRP-labeled for chemiluminescence. An intensified CCD camera allowed acquisition of the luminescent signal. After only 10 min of photon accumulation, on cell line smears as well as on serial tissue sections, chemiluminescence gave comparable results to those obtained by a 3-week exposure for 35S autoradiography. A quantitative approach on cervical biopsy specimens confirmed this similar level of sensitivity by measuring the area of 35S- or chemiluminescence-stained nuclei. Our results indicate that chemiluminescence is a credible and perfectible alternative to radioisotopes for in situ detection of nucleic acids by hybridization.

Ultrastructural localization of the major components of the extracellular matrix in normal rat nerve.

In this study we determined the ultrastructural distribution of the various components of the extracellular matrix (laminin, fibronectin, Type I, III, and IV collagens) of the normal peripheral nerve in adult rat. The localization of these macromolecules was investigated in basement membranes as well as in different areas of epi-, peri-, and endoneurium, by use of a pre-embedding immunoperoxidase method.

Steroidogenic adrenocortical cells synthesize alpha 2-macroglobulin in vitro, not in vivo.

We previously identified alpha 2-macroglobulin as the major protein secreted by primary cultures of adrenocortical cells. We report here that in the adrenal gland, the distribution of alpha 2-macroglobulin in the adrenocortical tissue is restricted to the endothelium of blood vessels and that no immunoreactivity is found in steroidogenic cells. A time course study revealed that freshly dissociated bovine adrenocortical cells were void of alpha 2-macroglobulin immunoreactivity whereas the proportion of alpha 2-macroglobulin-positive cells reached more than two-thirds of the population between day 4 and day 7 of culture. Double immunoenzymatic labeling of 6-day-old cultures revealed a co-localization of alpha 2-macroglobulin and the steroidogenic enzyme P-450SCC. Treatment of 5-day-old cultures (expressing alpha 2-macroglobulin) for 24 h by either ACTH (10(-9)-10(-6) M) or alpha 2-macroglobulin (2.5 mg/ml) resulted in a marked decrease of the expression of alpha 2-macroglobulin. These data indicate that ACTH and plasmatic alpha 2-macroglobulin could physiologically repress alpha 2-macroglobulin expression in the adrenal cortex in vivo.

Contribution of apoptosis to the phenotypic changes of adrenocortical cells in primary culture.

The aim of this study was to evaluate the occurrence and physiological consequences of apoptosis in primary cultures of bovine adrenocortical cells (of fasciculata-reticularis origin). Under ACTH-free culture conditions, we observed apoptotic cells in the cell layer and the accumulation of apoptotic bodies in the culture medium. These were hardly detectable in ACTH-supplemented cultures. Under ACTH-free conditions, the DNA content of apoptotic bodies collected over 48 h represented up to 10-15% of that of the cell layer at the onset of the culture (as compared to 3% in ACTH-supplemented cultures). Past the fourth day of culture in the absence of ACTh, most cells lacked several markers of their originating fasciculata-reticularis phenotype and progressively evolved to an undifferentiated phenotype. The vast majority of the apoptotic bodies released during the first 4 days of culture were immunoreactive for P450 17 alpha. Inversely, during the same period of time, the proliferating cells (PCNA-positive) did not appear to express P450 17 alpha. Therefore, apoptosis could contribute, together with dedifferentiation, to the phenotype shift observed in ACTH-depleted cultures of adrenal fasciculata-reticularis cells. These observations also characterize this endocrine cell system as an in vitro model for the study of hormone-repressed apoptosis.

Medullary thyroid carcinoma: search for histological predictors of survival (109 proband cases analysis).

A group of 13 pathologists belonging to the French Calcitonin Tumor Study Group (GETC: Groupe d'Etude des Tumeurs à Calcitonine) examined the histological slides and medical records of 109 proband cases of medullary thyroid carcinoma (MTC) diagnosed on clinical features. The cases belonged to the various forms of the disease (80 sporadic and 29 familial MTC). The aim of the study was to detect histological predictors for survival by comparing morphological data from patients killed by the disease versus the others. Twenty-seven histological parameters were considered, including cellular heterogeneity, shape of the cells, and cytoplasmic characteristics. Other parameters such as sex, age, and phenotype of the disease were also studied. First, predictive parameters of interest on survival function were selected by univariate analysis (Mantel-Cox test). Then, the extracted parameters were tested in a multifactorial analysis using the Cox's forward stepping proportional hazard model. Five parameters were significantly associated with a lower survival function: presence of necrosis in the tumor (P = .001), squamous pattern (P = .002), age over 45 years (P = .004), presence of oxyphil cells in the tumor and absence of cells with intermediate cytoplasm (P = .025), less than 50% of calcitonin immunoreactive cells in the tumor (P = .04).

Graves-Basedow disease goiter: a model of Bax-Bcl2 regulated apoptosis.

This study demonstrates the involvement of a Bax-Bcl2-dependent apoptotic process in Graves-Basedow thyroid disease, a pathological condition known for its spontaneously oscillating evolution. A continuous series of 86 cases of surgically treated Graves' thyroid was evaluated for apoptotic cell content identified by histological criteria and confirmed by terminal desoxynucleotidyl transferase-mediated desoxyuridine triphosphate nick end-labeling (TUNEL). A significant correlation was found between tissue features of Graves' disease (epithelial hyperplasia, cellular hypertrophy, colloid content) and the amount of apoptotic cells. No correlation was found with lymphocytic infiltrates. Significantly, 11 cases (about 12% of the series) with high-level apoptosis displayed the typical features of active Graves' disease over all tissue sections. In contrast, cases with no detectable apoptosis exhibited regressive tissue features of Graves' disease. An intermediate group of cases was characterized by tissue heterogeneity with hyperactive foci, rich in apoptosis, alternating with regressive areas lacking apoptosis. In this group the participation of apoptosis to the remodeling of Graves' thyroid parenchyma, in a tight balance with cell proliferation, was best illustrated. Moreover, the thyroid follicle by accumulating apoptotic cells and bodies, allowed a tentative chronological ordering of apoptosis steps in correlation with Bax-Bcl2 tissue distribution and cellular pattern. Our observations suggest that the initiation of apoptosis corresponds to a loss of cellular cohesion, a drop in Bcl2 expression, and a delocalization of Bax from a putative Golgi storage location to a mitochondrial distribution.

Importance of DNA fragmentation in apoptosis with regard to TUNEL specificity.

In the absence of a universal specific molecular tracer of apoptosis, structural DNA alterations provide the basis of labeling systems: double-strand fragmentation for TUNEL (terminal transferase-mediated dUTP nick end-labeling), denaturation for poly (A) in situ hybridization, immunogenicity of single strand DNA, all methods which imply limited specificity due to the unavoidable presence of DNA breaks in virtually all cells. Thus, TUNEL application has been restrained to a narrow spectrum of sample conditions which has limited, in particular, retrospective surveys and apoptotic nuclei-protein double labelings. In the apoptotic nucleus two main obstacles intervene between TUNEL reagents and their targets: DNA hypercondensation and proteins around DNA. The former increases in the course of apoptosis and both are worsened by crosslinking and precipitating fixatives. This point out that TUNEL is an ambitious approach whose target, apoptotic DNA breaks, is less accessible than breaks occurring in non-apoptotic less compacted DNA. However, TUNEL has an advantage: the far greater degree of apoptotic DNA fragmentation. How to obtain a frank differential staining between apoptotic and non-apoptotic DNA? It appears that the answer relies on the pretreatment step and not in modifying the TUNEL staining protocol, which is optimal. Adapted pretreatments are able to circumvent accessibility obstacles and to extend TUNEL applicability to the most demanding conditions, those of archived tissue samples and of TUNEL--protein double labelings.

Evaluation of 29 monoclonal and polyclonal antibodies used in the diagnosis of pituitary adenomas. A collaborative study from pathologists of the Club Français de l'Hypophyse.

Fifteen polyclonal antibodies (pAbs) and 14 monoclonal antibodies (mAbs) directed against hGH, hPRL, beta hFSH, beta hLH, beta hTSH and alpha-subunit were assessed by five different laboratories on normal and adenomatous pituitary tissues. This study aims at providing pathologists with a selected panel of antisera suitable for diagnosis, and appreciating the interest of the recently introduced mAbs. All the anti-hGH Abs proved to be specific (3 pAbs and 4 mAbs); three mAb out of four gave a few false-negative reactions. Three out of six polyclonal anti-hPRL showed cross-reactivity with hGH; anti-hPRL mAbs gave a strong staining with no false-negativity detected so far. MAbs proved to be more efficient for detecting glycoprotein hormones and alpha subunit than pAbs, which, in several cases, gave widespread cross-reactivity. This lack of specificity could explain the noticeable discrepancies reported so far in the appraisal of gonadotropic and somatoprolactinic adenomas.

Intracranial plasma cell granuloma: a report of four cases.

Inflammatory pseudotumors (IPT) are rare lesions composed of inflammatory cells admixed with collagen tissue. Although IPT are ubiquitous, intracranial locations are rare. In this study, four intracranial IPT of the plasma-cell-granuloma (PCG) type are reported. Four patients presented with lesions located, respectively, in the right cavernous sinus, the left cavernous sinus with extension to the tentorium cerebelli, the vermis cerebelli, and the pituitary stalk. All patients were operated on, but complete resection could not be achieved in cases 1 and 2. Follow-up was favorable in all cases, although case 1 still complained of headaches 2 years after operation. All cases were studied on histologic and immunohistochemical bases, and ultrastructural analysis was performed on two cases. In cases 1, 2, and 4, IPT were made up of plasma cells admixed with lymphocytes and rare histiocytes in a fibrous tissue-the density of which varied from case to case. In case 3, the mass was composed of plasma cells associated with numerous foamy histiocytes and polymorphonuclear cells. No light chain restriction could be demonstrated when immunohistochemistry was performed, and ultrastructural study did not disclose features reminiscent of meningioma or histiocytosis X. Intracranial IPT should not be confused with other diseases such as meningioma, lymphoproliferative disorders, or histiocytosis X. Although intracranial locations are much rarer than pulmonary ones, histology is identical in both sites and shows different patterns in its evolution. This is in agreement with the inflammatory origin of this lesion.

[112 cases of sporadic and genetically determined pheochromocytoma: a comparative pathologic study]. / 112 cas de phéochromocytomes sporadiques et génétiquement déterminés: Etude anatomo-pathologique comparative.

The aim of this study was to compare 64 genetically determined pheochromocytomas (PH) (49 MEN IIa, 3 MEN IIb, 6 Von Recklinghausen diseases, 1 von Hippel-Lindau disease, 5 familial pheochromocytomas) and 48 sporadic PH. Genetically determined PH were more often observed among men and more frequently bilateral and multicentric than sporadic PH. Sporadic tumors had more often adrenal capsular invasion, necrosis and pseudocysts. Genetically determined PH were more differentiated with an insular pattern, hyaline globules and a higher percentage of polyhedric cells. Sporadic tumors were less differentiated with more frequently a diffuse pattern and small cells. Adrenal medullar hyperplasia was significantly associated with genetically determined PH. Adrenal cortical hyperplasia was not associated with a particular type of PH. The PS100 and chromogranin immunodetection was equivalent in both groups.

[Role of hormonal exploration in avoiding of the failures of anterior pituitary gland surgery in Cushing disease]. / Rôle de l'exploration hormonale dans l'esquive des échecs de la chirurgie anté-hypophysaire de la maladie de Cushing.

Patients with Cushing's disease are not cured by transsphenoidal microsurgery in about 30% of the cases. Beside the problem of invasive macroadenomas, these failures are due either to diagnostic errors, or to very small microadenomas that could no be found. Positive diagnosis of hypercortisolism is quite straightforward and the problem is sensitivity rather than specificity. Primary adrenocortical hypercortisolism should not be mistaken. Depression-related hypercortisolism can be difficult to distinguish from Cushing disease: most cases are recognized after clinical story and CRF stimulation test. Ectopic ACTH secretion by a carcinoid tumor represents at least 8% of ACTH-dependant hypercortisolism. It cannot be reliably distinguished from corticotroph microadenoma by either classical dynamic tests or anterior pituitary imaging. However measurements of ACTH in the inferior petrosal sinus under basal condition and CRF stimulation allow the diagnosis of central or peripheral ACTH secretion with a quasi 100% sensitivity and specificity. In contrast this technique is of poor help for the diagnosis of lateralization of corticotroph microadenomas, for which it gives erroneous results in 25 to 50% of the cases. Rapid intraoperative measurement of ACTH in peripituitary blood seems a more reliable approach. In our series it gave correct results in 11 out of 12 cases. In 1995 hormonal exploration of Cushing disease should limit the failures of anterior pituitary surgery to the cases of invasive macroadenomas that cannot be completely removed.

[Gonadotropic pituitary adenoma and pregnancy. Value of bromocriptine]. / Grossesse chez une femme traitée pour adénome gonadotrope. Intérêt de la bromocriptine.

The authors report a case of pituitary adenoma with positive immunospecific staining for beta-FSH and serum alpha subunit. The tumour was revealed by an amenorrhoea-galactorrhoea syndrome with hyperprolactinaemia and without increase in serum gonadotrophin values. Pregnancy occurred during bromocriptine treatment which made it possible to control an hemianopsia developed in the fourth month of gestation. The adenoma was successfully removed a few months later.