Somatic in frame deletions not involving juxtamembranous cysteine residues strongly activate the RET proto-oncogene.

Somatic RET mutations have been identified in a variable proportion (about 30-70%) of sporadic Medullary Thyroid Carcinoma (MTC) cases. They are represented by the Met918Thr substitution (exon 16) typical of Multiple Endocrine Neoplasia type 2B (MEN2B) and, to a lesser extent, by nucleotide changes occurring at one of five critical cysteine residues (exons 10 and 11) typical of MEN type 2A (MEN2A). An in vitro transforming activity has already been demonstrated for these mutations. A few different MTC somatic mutations have been reported so far whose biological activity has still to be tested. In this paper we report the identification, in two MTC tumor samples, of two interstitial deletions of 48 bp and 6 bp occurred in exons 10 and 11 respectively. Both were somatic heterozygous in frame mutations, not involving any cysteine residue. Moreover, the expression of a full length RET cDNA carrying one of the two deletions demonstrated a strong transforming capacity in NIH3T3 cells.

Monoclonal antibody immunocytochemistry: novel method extending usefulness of monoclonal antibodies for antigen visualization.

We describe a novel procedure combining the multiple-site reactivity of polyclonal antibodies with the defined single epitope-specificity of monoclonal antibodies. The method is based on previous findings that IgG molecules often only react with tissue-bound antigens with one of their two antigen-combining sites; thus, the remaining site is free to bind subsequently added antigen. In the procedure devised, such (undenatured) antigen is subsequently detected by a specific monoclonal antibody and the reaction is finally revealed by immunogold-silver staining. Antibody subpopulations to contaminating antigens may well be present in the polyclonal antiserum and may well bind first to tissue and then to the corresponding contaminants in the crude antigen preparation applied as second layer. Such contaminants will, however, not react with the monoclonal antibody and will therefore not be immunocytochemically detected. The method has been evaluated with one antigen which cannot be detected by monoclonal antibodies in paraffin sections (glial fibrillar acidic protein) and with another antigen (human chorionic gonadotropin) which can only be detected by the monoclonal antibody when occurring in high concentrations. In both cases the procedure resulted in strong specific staining of the antigens with no background.

Proopiomelanocortin producing cells of spleen: increase after transplantation with opioid-peptide producing Ehrlich ascites tumor cells.

Proopiomelanocortin (POMC) peptides are produced by many cell systems, including a population of macrophage-like cells in mouse spleen. After transplantation of mice with Ehrlich ascites tumor cells, the number of POMC producing spleen cells increase up to 10-fold by 5 to 6 days. The POMC peptides produced by these cells increase even more, as evidenced by radioimmunoassay. Thus, these data indicate both proliferation of splenic POMC cells and increased production of POMC peptides per cell after tumor challenge. Characterization of the peptides by sequence-specific radioimmunoassays and high performance liquid chromatography documents the presence of both ACTH(1-39) and of ACTH(1-14) in these cells. These peptides have multifacetted effects on immune parameters and may exhibit a general antiinflammatory action, partly mediated through inhibition of interleukin 1-stimulated events. The tumor cells themselves do not produce POMC peptides, but display met- and leu-enkephalin immunoreactivity. Also cultured tumor cells display such immunoreactivity, indicating endogenous production of opioid peptides. The opioid peptides of the tumor cells may both affect host immune defenses and play intratumoral autocrine or paracrine roles.

Proprotein convertases (PC1/PC3 and PC2) in normal and neoplastic human tissues: their use as markers of neuroendocrine differentiation.

By immunocytochemistry and immunoblotting, we examined normal and neoplastic human tissues with polyclonal antibodies raised against selected peptide regions of proprotein convertase-2 and -3 (PC2 and PC3), two proteases that have been shown to selectively cleave neuroendocrine precursor molecules at pairs of basic residues. Immunoreactivity for both enzymes was detected in neuroendocrine cells of pituitary, gut, pancreas, thyroid, and adrenals and in tumors thereof, but was absent in thyroid follicular cells, parathyroids, adrenal cortex, testes, and a number of nonneuroendocrine tissues, both normal and tumorous. Although both PCs were virtually universal concomitants of the neuroendocrine system, cells with a neural phenotype (e.g. pheochromocytes and Merkel cells) predominantly contained PC2, whereas classic endocrine cells contained mostly PC3. PC3 immunoreactive cells were abundant all along the gastrointestinal tract, whereas PC2 was highly expressed only in the pyloric antrum and proximal third of duodenum. Double immunostaining experiments revealed colocalization of PC3 with virtually all gastrointestinal peptides, whereas PC2 immunoreactivity was mostly expressed in gastrin, cholecystokinin, and somatostatin cells. Noticeably, the proportion of glucagon-producing cells immunoreactive for PC3 was high in the gut and low in pancreatic islets and glucagonomas, whereas the reverse occurred for PC2. At the ultrastructural level, immunostaining was confined to the mature dense core granules, the site of storage of granins and peptide hormones. With the exception of parathyroid cells, PC2 and/or PC3 expression correlated with the occurrence of granins, canonical markers of the secretory granules. Immunoblotting experiments confirmed the identity of the immunocytochemical reactivities. It is concluded that PC2 and PC3 are highly sensitive markers of neuroendocrine differentiation and have distinct distribution patterns, and that antibodies to these enzymes may play an important role in the analysis of tumors.

Mucinous carcinoma of the breast. A clinicopathologic, histochemical, and immunocytochemical study with special reference to neuroendocrine differentiation.

We studied the clinical, histologic, histochemical, and immunocytochemical characteristics of 61 mucinous tumors (38 pure, 23 mixed) retrieved from a consecutive series of 1,689 infiltrating carcinomas of the female breast. The only statistically significant predictors of favorable survival were histologic (pure) type coupled with the absence of axillary lymph node metastases. Other factors, including classification into A and B types according to Capella et al., and neuroendocrine status, as assessed by the presence of argyrophilia, granins, neuron-specific enolase (NSE), and synaptophysin (SYN),-all had no influence on survival. Argyrophilic cells were found in 16 pure mucinous tumors (42%) and in the mucinous component of four mixed tumors (17%). Granin (chromogranin A or B), NSE, and SYN immunoreactivities were demonstrated in all the argyrophilic tumors. We also found NSE- and SYN-immunoreactive cells in 31 of 41 and 16 of 41 nonargyrophilic (granin-unreactive) mucinous tumors, which supports the view that mucinous carcinomas of the breast as a whole are neuroendocrine-programmed tumors.

Argyrophilic carcinoma of the male breast. A neuroendocrine tumor containing predominantly chromogranin B (secretogranin I).

Argyrophilic tumors were diagnosed in 28 of 134 (20.8%) consecutive male patients who had a carcinoma of the breast removed between 1961 and 1990. Histologically, most argyrophilic tumors showed uniform cellularity and prevalent expansive growth. Ultrastructural observation disclosed the presence of electron-dense cored granules in the cytoplasm of the tumor cells. By immunocytochemistry, 17 of 28 argyrophilic tumors (60.7%) contained chromogranin B (secretogranin I)-immunoreactive cells, whereas chromogranin A was present in four of these 17 tumors only (14.2%). Immunoblotting studies showed chromogranin B immunoreactivity similar to that found in normal neuroendocrine cells. Despite these findings, which would argue for a distinct morphologic and immunochemical entity, no statistically significant differences between argyrophilic and common male breast carcinomas were found when a number of clinicopathologic features and relapse-free survival were considered.

Argyrophilia and granin (chromogranin/secretogranin) expression in female breast carcinomas. Their relationship to survival and other disease parameters.

Ninety-one tumors (5.6%) containing argyrophilic cells were identified in a series of 1,628 consecutive primary breast carcinomas diagnosed between 1968 and 1972 at the Istituto Nazionale Tumori, Milan, Italy. Histological evaluation of these argyrophilic tumors showed the presence, either throughout the whole tumor mass (pure form) or in some areas (mixed form), of distinctive though not pathognomonic cellular features. Immunocytochemistry revealed the presence of chromogranin A or chromogranin B (secretogranin I) immunoreactivity in 86% of these argyrophilic carcinomas and of neuron-specific enolase (NSE) immunoreactivity in all of them. The three neuroendocrine markers were also immunolocalized at the ultrastructural level in the dense-core granules (granins) and the cytoplasmic matrix (NSE). Immunoblotting studies confirmed the chromogranin A and B and NSE immunoreactivities and documented the presence of secretogranin II. We also studied the relation of the histologic, histochemical, and immunocytochemical features to prognosis. There was no significant correlation between argyrophilia and such clinical parameters as age, menopausal status, tumor size, and overall survival; however, the pure form of argyrophilic tumors had a significant association with less frequent lymph node involvement and a low histologic grade.

Nonspecific immunocytochemical reactions with certain neurohormonal peptides and basic peptide sequences.

Immunocytochemical staining experiments on filter paper or nitrocellulose models reveal that many, but not all, neurohormonal peptides, as well as poly-L-lysine, strongly bind a number of labeled reporter molecules, including colloidal gold- or peroxidase-labeled IgG, protein A, streptavidin, and albumin. Peptides displaying this type of (nonspecific) binding are basic; they include ACTH, VIP, opioid peptides, and poly-L-lysine. Pre-absorption of labeled probes with excess ACTH[1-24] or poly-L-lysine abolishes or greatly reduces binding not only to the homologous but also to the heterologous peptides tested. A number of cell types previously reported to display nonspecific immunoglobulin binding contain one or several of the basic neurohormonal peptides shown to display nonspecific absorption of labeled IgG, protein A, streptavidin, and albumin. This nonspecific absorption is reversed neither by high salt nor high pH conditions, nor by a number of detergents and blocking proteins. One dynorphin antiserum also displays nonspecific binding to the peptides as well as to pancreatic glucagon cells, and this nonspecific staining can be blocked by basic peptide pre-absorption (whether homologous or heterologous). These results suggest a need for caution when immunocytochemical studies of a number of basic polypeptides are interpreted, and also suggest the inclusion of novel control procedures in immunocytochemistry.

Non-sulphated cholecystokinin in human medullary thyroid carcinomas.

The expression of gastrin/cholecystokinin (CCK) peptides and their precursors was examined in 16 medullary carcinomas of the human thyroid. Measurements with libraries of sequence-specific radioimmunoassays before and after enzymatic cleavage of extracts and chromatographic fractions showed that the carcinomas contained 1.7 pmol carboxyamidated CCK/g tissue (median; range 0.6-21.8 pmol/g), 0.9 pmol glycine-extended precursor/g (median; range less than 0.2-2.3 pmol/g) and 2.3 pmol further COOH-terminal-extended proCCK/g (median; range 0.9-6.2 pmol/g). Neither carboxyamidated gastrins nor any progastrins could be measured. Gel and reverse-phase chromatography revealed only small molecular forms, i.e. greater than 90% of the amidated immunoreactivity eluted like non-sulphated CCK-8 or CCK-7. The results show that human medullary thyroid carcinomas synthesize CCK peptides. The predominance of non-sulphated CCK is unusual. Taken together with earlier observations from dogs and pigs, our results raise the possibility that small non-sulphated CCK peptides modulate thyroid C-cell secretion in an autocrine manner.

Immunocytochemical localization and identification of prosomatostatin gene products in medullary carcinoma of human thyroid gland.

Thirty-three cases of histologically proven calcitonin-positive medullary thyroid carcinoma were studied immunocytochemically for the occurrence of prosomatostatin-related peptides. Positive cells, identified with a panel of antisera raised against four different regions of the prosomatostatin molecule, were found in 100% of the tumors. Most but not all somatostatin-positive cells were also immunoreactive for calcitonin. Notably, seven patients harboring somatostatin-rich tumors revealed a more favorable clinical course. The results (1) indicate that somatostatin production is a universal concomitant of thyroid medullary carcinoma, (2) suggest that these cells are likely to produce a somatostatin precursor molecule similar to mammalian prosomatostatin, and (3) imply that somatostatin-reactive cells may have as yet unknown roles in these tumors, possibly in the realm of paracrine and autocrine regulation of cell growth.

Differentiation in paediatric peripheral primitive neuroectodermal tumours of bone. A critical contribution to its assessment.

Differentiation was studied in 73 paediatric peripheral primitive neurorectodermal tumours (pPNETs) of bone observed during 1974 through 1992. The presence of rosettes, pseudorosettes, and/or a rosette-like arrangement of tumour cells (the morphological neural marker, MNM) occurred in 29% of these cases. NSE and N-CAM were expressed by nearly all tumours; synaptophysin was present in 30% of cases, not significantly associated with the MNM status. Neuroendocrine (NE) markers were present in 25% (chromogranin B, secretogranin II) to 40% (chromogranin A, 7B2 protein) of cases. Focal expression of cytokeratins, S100 protein and/or desmin was also noted in a minority of cases. In univariate statistical analysis, only the presence of MNM conferred a significantly higher (about twofold) risk of death than its absence. This study demonstrates the occurrence of at least one immunocytochemical N and/or NE differentiation marker in all pPNETs of bone and a focal expression of cytokeratins, S100 protein and/or desmin in a minority of cases. Synaptophysin and MNM were present each in less than 1/3 of the cases, and no association was noted between them. Statistical analyses highlighted the prognostic role of MNM per se and discourage the sole use of immunocytochemistry in the assessment of neuroectodermal differentiation for prognostic purposes in paediatric pPNETs of bone.

Functioning human insulinomas. An immunohistochemical analysis of intracellular insulin processing.

Sixty-seven insulinomas were investigated by immunohistochemistry using site-directed antibodies against insulin, proinsulin, chromogranin A, HISL-19, and four proteins directly or indirectly involved in the proteolytic processing of proinsulin: the prohormone convertases PC2 and PC3, carboxypeptidase H (CPH) and 7B2. Results were expressed in a six-grade score according to the frequency of immunoreactive tumour cells. Insulin was expressed by all tumours, appearing in either a diffuse or a polarized pattern and being detected in more than 30% of tumour cells in all cases but three. Proinsulin was also expressed in all tumours, with more than 50% of tumour cells immunoreactive in all cases but 5. It was consistently localized in the Golgi apparatus. In about half the cases, moreover, it also showed diffuse cytoplasmic staining, usually with a very sparse distribution. Trabecular and solid insulinomas did not present specific, homogeneous patterns of insulin immunostaining. However, insulin immunoreactivity was much more abundant in trabecular than in solid neoplasms, being present in virtually all tumour cells (score 6) in 50% and 8% of cases, respectively. Virtually all insulinomas expressed PC2, PC3, CPH and 7B2, usually in 30-100% of tumour cells, with a frequency significantly related to that of insulin. However, detection of PC2 and 7B2 was slightly less frequent than that of PC3 and CPH. In consecutive sections these proteins were found to be mostly co-localized with insulin and chromogranin A but not with proinsulin. They were heavily expressed in all 10 tumours with more than 10% of cells showing cytoplasmic proinsulin immunoreactivity, indicating that the leakage of proinsulin from the Golgi compartment is not associated with faulty expression of converting enzymes and possibly reflects a saturated processing capacity. HISL-19 immunoreactivity was found in both Golgi apparatus and insulin stores, indicating that the relevant antigen is different from all other proteins investigated. These results do not support a defect in expression or localization of proinsulin-processing enzymes in most insulinomas.

Immunocytochemical detection and characterization of intrahepatic human pancreatic islets after combined liver-islet allotransplantation.

The unique availability of an explanted liver-islet allograft, removed for primary nonfunction of the liver, led us to evaluate distribution and phenotype of exocrine and endocrine components of the pancreatic graft. Immunocytochemistry was used to map patterns of gene products for islet hormones, proprotein processing enzymes, panneuroendocrine markers, and pancreatic exocrine markers. When compared with age-matched control pancreases, insulin-, glucagon-, somatostatin-, and pancreatic polypeptide-producing cells were similarly represented and distributed within the grafted islet. We also demonstrate that the intrahepatic transplanted islets retained the enzyme machinery able to process the hormone precursors into bioactive fragments. In the clinical setting, this resulted in an immediate functioning of the graft and insulin-independence of the patient one month after transplantation. The purity in islets, as assessed by immunocytochemistry with antibodies to tissue constituents of endocrine and exocrine lineages, was around 40%. Despite the massive intraportal presence of pancreatic acinar tissue, no signs or symptoms attributable to ectopic hypersecretion of exocrine enzymes occurred. In fact, when tested with antibodies to such enzymes, low levels of immunoreactivity were observed in the grafted acinar cells.

Immunocytochemical localization and identification of members of the pancreatic polypeptide (PP)-fold family in human thyroid C cells and medullary carcinomas.

An increasing number of regulatory peptides not coded by the calcitonin genes are known to occur in the thyroid C cells. We have now carried out light and electron microscopic immunocytochemical analyses on specimens of normal human thyroids and medullary carcinomas to establish the occurrence of members of the PP-PYY-NPY family in the C cell system. By means of site-directed immunocytochemistry we provide the first evidence that a molecule closely related to proNPY is present in normal and pathologic C cells, and is co-stored with calcitonin in the cytoplasmic dense-core granules. Preliminary observations also suggest that high levels of expression of NPY-gene products help to define a subset of tumours with a less aggressive behaviour.

Prognostic impact of thyroid lymphocytic infiltration in patients with medullary thyroid carcinoma.

A retrospective search for lymphocytic infiltration of the thyroid was performed on archival paraffin specimens from 95 cases of thyroid medullary carcinoma observed at a single institution during a 30-year period. A mild lymphocytic infiltration of the nonneoplastic thyroid tissue, mainly concentrated at the edge of the tumor mass, was observed in 33 cases, while in a further 24 cases a moderate to marked lymphocytic infiltration, resembling that seen in chronic lymphocytic thyroiditis, was homogeneously distributed all over the gland, with no apparent relationship to the site of the primary tumor. Virtually no lymphocytic infiltration was detected inside of any of the tumors. The presence of chronic lymphocytic thyroiditis seemed to be a characteristic of the host rather than a tumor-induced event. Statistical univariate analysis of relapse-free survival and overall survival showed that lack of chronic lymphocytic thyroiditis marked those patients with an increased risk of disease recurrence and death. However, the observed risk for survival was statistically nonsignificant, whereas that for disease recurrence was significant and remained in a multivariate model of statistical analysis.

Silver enhancement of nickel-diaminobenzidine as applied to single and double immunoperoxidase staining.

A reliable and practical method is proposed for increasing sensitivity and detection efficiency of immunocytochemical techniques, based on silver enhancement of the nickel-diaminobenzidine product of the peroxidase reaction. The procedure produces a strong signal at the site of the end product of the peroxidase reaction which is visible as black grains at the light microscopic level. The method has been used to detect peroxidase labeled probes in immunocytochemical tissue preparations and blotting assays and is ideal for the purposes of double staining and photographic documentation.

Well-differentiated neuroendocrine carcinoma of the lung: a clinicopathologic and ultrastructural study of 10 cases.

The clinico-pathologic characteristics of 10 resected pulmonary tumors, which proved to be well-differentiated neuroendocrine carcinomas (WDNC) on the basis of light microscopic, immunocytochemical, ultrastructural and immunoelectron microscopic investigations, were evaluated. The tumors showed a wide spectrum of histologic features that could be referred to three basic patterns: 1) a carcinoid-like pattern; 2) an organoid pattern characterized by palisading cells at the edge of cellular areas, and 3) a prevalent adenocarcinoma-like pattern. The second pattern was the most distinct even though it often mimicked the small cell/large cell subtype of small cell carcinoma (SCC) owing to its association with marked atypia and poor differentiation. All but one of the patients were males and smokers. The mean age was 58 years. Half of the tumors were centrally located including those showing the adenocarcinoma-like pattern. Disease-free and overall survival and type of tumor dissemination in four patients were similar to those of SCC. Five evaluable patients were alive and disease-free after a mean follow-up of 74 months. Two of these were initially diagnosed as SCC. We conclude that, because of its impact on prognosis, the diagnosis of WDNC appears to be relevant although other factors able to adversely affect the clinical course remain undefined.

Malignant pheochromocytoma in multiple endocrine neoplasia type 2B syndrome. Case report and review of the literature.

A malignant behavior (i.e., distant metastatic spread) has been recorded in 3-4% pheochromocytomas occurring in the context of multiple endocrine neoplasia type 2A syndrome, but has never been documented in patients with the type 2B form. In this report we describe a case of malignant pheochromocytoma arising in the latter syndrome setting. The patient, a white young male, had the full-blown syndrome, including multicentric, bilateral medullary thyroid carcinoma metastatic to regional lymph nodes, mucosal neuromas, digestive ganglioneuromatosis, marfanoid habitus, and bumpy lips. Three and a half years after surgical resection of an apparently benign adrenal pheochromocytoma he developed widespread osseous metastases. The presence of hypertensive crises and high urinary catecholamine excretion rates, coupled to moderate hypercalcitoninemia, normal circulating carcinoembryonic antigen levels, negative whole-body 99mTc-(V) dimercaptosuccinic acid scan, and absence of neck or mediastinal disease by magnetic resonance imaging, proved that the metastases were from his previous adrenal and not thyroid tumor. Furthermore, since the bone metastases strongly accumulated 131I-metaiodobenzylguanidine, several courses of the radiocompound were given, which resulted in an objective, though partial, tumor regression.

Prognostic value of clinicopathologic characteristics including neuroectodermal differentiation in osseous Ewing's sarcoma family of tumors in children.

AIMS AND BACKGROUND: The aim of the present study was to determine the relationship between clinico-pathologic parameters, including neuroectodermal differentiation, and their impact on survival in a series of pediatric patients with osseous tumors of the Ewing's sarcoma family admitted to the Pediatric Department of the Istituto Nazionale Tumori of Milan. METHODS: Seventy-three patients were enrolled. The variables analyzed were sex, age, site of primary tumor, serum lactate dehydrogenase (LDH) level at diagnosis, involvement of periosseous soft tissues by primary tumor, presence of metastatic disease, status of disease after the treatment plan, as well as the presence of mitoses, morphologic and immunocytochemical neural markers, and neuroendocrine markers in the primary tumor. RESULTS: Neural and neuroendocrine markers were not significantly associated with any of the other parameters. In the univariate analysis, significant risk factors related to unfavorable outcome were elevated LDH, metastatic disease, lack of complete remission after treatment, presence of mitoses and of morphological neural markers; immunocytochemical neural and neuroendocrine markers lacked prognostic value. In the multivariate analysis, only LDH levels and the status of disease following the treatment were retained. CONCLUSIONS: LDH level at diagnosis might be a useful marker to identify different risk levels; neuroectodermal differentiation might have no clear-cut impact on the clinical management of osseous Ewing's sarcoma family of tumors.