Lack of RUNX3 regulation in human gastric cancer.

It has been proposed that the transcription factor RUNX3 is the product of a gastric tumour suppressor gene. We examined RUNX3 expression in gastric biopsies from 105 patients with different histological presentations. Surprisingly, immunohistochemical staining detected RUNX3 protein expression only in infiltrating leukocytes but not in the gastric epithelium. Using laser capture microdissection and quantitative reverse transcription-polymerase chain reaction, we confirmed that the level of RUNX3 mRNA expression in the gastric epithelium was very low and was influenced neither by H. pylori infection nor by neoplastic transformation. Instead, RUNX3 was highly expressed in the gastric stroma and the level of expression correlated with the magnitude of H. pylori-induced gastric inflammation. The low level of RUNX3 expression in gastric epithelium and the absence of downregulation in gastric cancer do not support the hypothesis that RUNX3 functions as a gastric tumour suppressor gene.

Free peritoneal tumour cells are an independent prognostic factor in curatively resected stage IB gastric carcinoma.

BACKGROUND: Several studies have shown that the cytological detection of free peritoneal tumour cells (FPTCs) in patients with gastric cancer indicates the presence of metastatic disease. The immunocytochemical detection of FPTCs, especially in early-stage tumours, has not been examined comprehensively. METHOD: Peritoneal lavage was performed in 351 patients before curative resection of a gastric carcinoma between 1987 and 2001, and an adequate sample was obtained from 346 patients. FPTCs were detected immunocytochemically using Ber-EP4 antibody. Median follow-up time was 70 months. RESULTS: FPTCs were detected in the lavage fluid of 74 patients (21.4 per cent) and correlated with increasing pathological tumour depth (pT) and lymph node (pN) status (P < 0.001). The 5-year overall survival of patients with FPTCs was significantly worse than that of patients without FPTCs (35 versus 71.9 per cent; P < 0.001). FPTCs were present in 14 (8.5 per cent) of 164 patients with stage IA or IB tumours. Although the detection of FPTCs had no prognostic significance for stage IA tumours, the presence of FPTCs in those with stage IB tumours was associated with a worse prognosis (P < 0.001). Multivariate analysis identified the presence of FPTCs as an independent prognostic factor in the whole cohort and in the stage IB subgroup. CONCLUSION: Detection of FPTCs is associated with poor prognosis even in patients with early-stage gastric cancer and should be used for risk-group stratification.

p53 Mutations in nasal natural killer/T-cell lymphoma from Mexico: association with large cell morphology and advanced disease.

Nasal NK/T-cell lymphoma is a unique form of lymphoma highly associated with Epstein-Barr virus, and with a characteristic geographic distribution. Recently, we showed that p53 is overexpressed in a high percentage of nasal NK/T-cell lymphomas. The aim of this study was to analyze the status of the p53 gene, and correlate it with the expression of p53 protein and its downstream target, the cyclin-dependent kinase inhibitor p21, in a series of 25 cases of well-characterized nasal NK/T-cell lymphoma from Mexico. The highly conserved exons 5 to 8 of the p53 gene were amplified by polymerase chain reaction and screened for mutations by denaturing high-pressure liquid chromatography. Abnormal polymerase chain reaction products detected by denaturing high-pressure liquid chromatography and additional selected cases were sequenced. In addition, the incidence of loss of heterozygosity at the p53 locus was analyzed in 12 cases. Of the 25 patients, 17 were male and 8 female (M:F ratio, 2.1:1), with a median age of 43 years (range, 21 to 93 years). Morphologically, most of the cases were composed of a mixture of medium-sized cells and large transformed cells (21 cases), and four cases were composed exclusively of large transformed cells. Three different groups determined by p53 gene status and expression of p53 protein were identified: group 1 was p53 +/p53 mutated (five cases, all with p53 missense mutations). Morphologically, three of the five cases were composed of large cells. All five cases revealed overexpression of p53 in the majority of the tumor cells with a mean of 86%. Unexpectedly, three of these cases also showed overexpression of p21. Four of the five patients presented with clinical stage IVB and died with disease. Group 2 was p53+/p53 wild-type (10 cases). Histologically, nine cases were of the mixed type, and one of the large cell type. The percentage of p53 overexpressing cells was lower than in the previous group with a mean of 23%. p21 was positive in 7 of the 10 cases. Six patients in this group presented with clinical stages I to II and four patients with advanced disease (stage III and IV). Five patients are alive 12 to 120 months later (mean, 24 months), three with no evidence of disease. Group 3 was p53-/p53 wild-type (10 cases). All cases showed mixed cell morphology. p21 was positive in 5 of 10 cases. Four patients presented with clinical stage I to II and six patients with advanced disease. Four patients are alive with no evidence of disease 9 to 60 months later (mean, 10 months). Overall, p53 mutations were present in 24% (5 of 21) of the evaluable cases, all of them overexpressing p53 in the majority of tumor cells. Cases with p53 mutations were associated with large cell morphology (P = 0.0162) and presented more often with advanced stage disease. Loss of heterozygosity at chromosome 17p was found only in 2 of the 12 (17%) cases investigated, both cases showed p53 mutations of the remaining allele. P21 overexpression (60% of cases) is frequent in nasal NK/T-cell lymphoma and seems to be independent of p53 gene status. The overexpression of p53 and p21, independent of p53 mutations, although as yet not clear, might be the result of Epstein-Barr virus infection, and warrants further investigation.

p27(Kip1) immunostaining for the differential diagnosis of small b-cell neoplasms in trephine bone marrow biopsies.

The distinction between mantle cell lymphoma (MCL) and other small B-cell non-Hodgkin lymphomas (NHL) is important because MCL has a more aggressive clinical course. In bone marrow (BM) biopsy specimens, this distinction can be particularly difficult. Although cyclin D1 immunostaining and molecular detection of the t(11;14) translocation are highly specific markers for MCL, they fail to detect a proportion of cases. We have recently described that MCL typically lacks detectable expression of the cyclin-dependent kinase inhibitor p27(kip1) protein by immunostaining, which is expressed at high levels in most small B-cell NHL inversely correlated to the proliferation rate. We therefore examined whether p27(kip1) immunostaining could be a useful adjunct for the differential diagnosis of small B-cell NHL infiltrates in the BM. Trephine BM biopsy specimens of 96 patients, including well-characterized MCL (19 cases), B-cell chronic lymphocytic leukemia (27 cases), follicular lymphoma (18 cases), hairy cell leukemia (22 cases), and marginal zone lymphoma (10 cases) as well as 10 reactive BM, including five with benign lymphoid aggregates were investigated. In addition, the presence of a t(11;14) translocation involving the major translocation cluster was studied by PCR in all MCL. All cases of B-cell chronic lymphocytic leukemia, follicular lymphoma, and marginal zone lymphoma revealed a strong p27(kip1) nuclear staining in the majority of neoplastic cells. Fourteen (78%) cases of MCL were p27(kip1)-negative in the tumor cells, whereas four cases revealed a weak nuclear positivity. Seventeen (77%) cases of hairy cell leukemia were also either completely negative for p27(kip1) or showed a faint positive staining in a minority of the neoplastic cells. Nine of 19 cases (47%) of MCL showed a bcl1 rearrangement involving the major translocation cluster region. These findings demonstrate that p27(kip1) immunostaining is a valuable additional marker for the differential diagnosis of small B-cell NHL infiltrates in BM biopsies. The reduction or lack of p27(kip1) protein expression in MCL, as well as in hairy cell leukemia, might be an important event in the pathogenesis of these disorders.

PCR analysis of IgH-gene rearrangements in small lymphoid infiltrates microdissected from sections of paraffin-embedded bone marrow biopsy specimens.

The differentiation of benign lymphoid infiltrates from nodular infiltrates of B-cell lymphoma is difficult in bone marrow (BM) biopsy specimens taken from patients with non-Hodgkin's lymphoma (NHL). We investigated whether the determination of clonality by polymerase chain reaction (PCR) analysis of the immunoglobulin heavy chain (IgH) genes could be of help for the distinction of benign and malignant lymphoid infiltrates. BM biopsy specimens of 28 patients were studied, comparing PCR of entire bone marrow sections with microdissected nodular lymphoid infiltrates. Patients were divided into 4 groups according to morphologic criteria: group 1 (n = 12), positive for B-NHL infiltration; group 2 (n = 5), suspicious for infiltration by known B-NHL; group 3 (n = 5), morphologically benign infiltrates in patients with B-NHL; group 4 (n = 6), benign lymphoid infiltrates in patients without history of B-NHL. PCR products were analyzed using polyacrylamide gels and a fragment length analysis system (Genescan). PCR of whole sections showed clonal amplification products in all cases of group 1 and 1 case of group 2. PCR analysis from microdissected nodular infiltrates showed the presence of a clonal B-cell population in 5 additional cases of groups 2 and 4. In 3 of these cases, clonal rearrangements of corresponding size were obtained from the primary lymphoma biopsy specimens. None of the cases of group 3 showed evidence of a clonal population with either technique. The results indicate that microdissection of small nodular lymphoid infiltrates from paraffin-BM sections increases the sensitivity of IgH gene rearrangement analysis. To avoid detection of biologically irrelevant clonal populations, comparison of PCR products obtained from the BM and the primary lymphoma biopsy is advisable.

The use of molecular biology in diagnosis and prognosis of gastric cancer.

The investigation of molecular and genetic changes in gastric cancer has brought new insights into the pathogenesis of the disease. Knowledge of the genetic abnormalities and altered molecules could be used for differential diagnosis in case of an unknown primary tumor, allows their evaluation as prognostic factors, and could open novel avenues for more specific clinical interventions. Clinically relevant molecules whose expression or structure is altered include the plasminogen activator and its inhibitor plasminogen activator inhibitor type 1, the cell cycle regulator cyclin E, epidermal growth factor, the apoptosis inhibitor bcl-2, the cell adhesion molecule E-cadherin, and the multifunctional protein beta-Catenin. In addition, genetic instability is commonly seen. Gene amplification and protein overexpression of the growth factor receptors c-erbB2 and K-sam may be prognostic factors for intestinal- and diffuse-type gastric cancer, respectively. There has long been evidence for a genetic predisposition to gastric cancer by epidemiological studies and case reports. Very recently, germ line mutations of E-cadherin have been identified that are responsible for a dominantly inherited from of diffuse-type gastric cancer and could be used to identify individuals that are at high risk. The clinical implications of the recent findings for diagnosis, prognosis, therapy, and risk assessment are discussed.

Detection of Epstein-Barr virus in cutaneous and lymph nodal anaplastic large cell lymphomas (Ki-1+).

Epstein-Barr virus (EBV) is a gamma DNA herpes virus which is thought to play a part in the pathogenesis of some non-Hodgkin's lymphomas in individuals with or without immunodeficiency. We investigated 16 lymph nodal and 12 cutaneous anaplastic large cell lymphomas (ALCLs) (Ki-1+), all of which were in patients without immunodeficiency, for the presence of EBV genomes. The highly sensitive polymerase chain reaction (PCR) technique was employed for detection of viral DNA in extracts from formalin-fixed, paraffin-embedded tissue sections. In addition, we performed radioactive and non-radioactive in situ hybridization (ISH) for localization of EBV at the single cell level. EBV-DNA was demonstrated by PCR in five cases of nodal ALCLs (31%). All cutaneous ALCLs were negative. EBV-encoded small nuclear RNAs (EBERs) could be identified by ISH in the tumour cells of one of the five EBV-DNA-positive patients. Our results further support the concept that EBV may be involved in the development of a proportion of nodal ALCLs, but not in cutaneous ALCLs.

Expression of gastrin-releasing peptide (human bombesin) gene in large cell undifferentiated carcinoma of the lung.

Production of the growth factor gastrin-releasing peptide (GRP) or human bombesin has been shown to be a feature of neuroendocrine tumours of the lung, particularly small cell carcinoma, and is possibly responsible for the characteristically rapid growth of this tumour. Large cell undifferentiated carcinoma of the lung (LCC) is also characterized by rapid growth and there is increasing evidence that some LCCs exhibit neuroendocrine differentiation. We therefore investigated GRP/bombesin immunoreactivity and the expression of GRP gene in ten LCCs. Histologically, all were composed of large cells with abundant cytoplasm, open nuclei, and prominent nucleoli, and there was no evidence of squamous, glandular, or neuroendocrine differentiation. At the ultrastructural level, most showed squamous or glandular differentiation but none contained neuroendocrine granules. None of the tumours showed immunoreactivity for GRP/bombesin but seven of the ten showed a focal hybridization signal when treated with 32P-labelled GRP cRNA probes, indicating the presence of GRP mRNA. This was confirmed by northern blot analysis. This study shows for the first time that GRP gene is expressed in LCC. The production of GRP may contribute to the aggressive behaviour of LCC.

Localization of thyrotropin-releasing hormone prohormone messenger ribonucleic acid in rat brain in situ hybridization.

We studied the distribution of pro- TRH mRNA in rat brain by in situ hybridization histochemistry using radiolabeled single stranded cRNA probes to confirm the hypothesis that the TRH precursor is distributed beyond regions that contain immunoreactive TRH. All regions of the central nervous system previously recognized to contain TRH showed hybridization. Hypophysiotropic neurons in the medial parvocellular division of the paraventricular nucleus showed more intense hybridization than anterior parvocellular division cells, suggesting regional differences in expression. In addition, regions not previously recognized to contain TRH in neuronal perikarya by immunocytochemistry showed specific hybridization for pro-TRH mRNA. These include cells in the olfactory bulbs, dorsal motor nucleus of the vagus, ventrolateral periaqueductal gray, reticular nucleus of the thalamus, and anterior commissural nucleus. Only a single hybridizing band was observed on Northern blots of RNA extracts of the periaqueductal gray and reticular nucleus, identical to that seen in extracts of the paraventricular nucleus. The appearance of pro-TRH mRNA in neurons not previously recognized to contain TRH but which contain the prohormone suggests that non-TRH peptides within the TRH precursor may be preferentially expressed in certain regions of the brain.

Immunohistochemical and histochemical markers of primary lung cancer, lung metastases, and pleural mesotheliomas.

Sections of primary lung carcinomas, lung metastases, mesotheliomas, and lung metastases of some rare mesenchymal tumors were incubated with different cytokeratin (CK), vimentin, desmin, and tissue polypeptide antigen (TPA) antibodies and with antibodies reactive with different hormones (ACTH, PTH, alpha-HCG, Calcitonin CT), CEA, carcinoma-associated antigen (CA1), secretory component (SC), neuron-specific enolase (NSE), alpha-1-antitrypsin (alpha-1-AT), lysozyme (lyso), and S-100 protein (S 100). CK antibodies derived from a 49 kD (reactive with simple epithelia [SE]) and a 67 kD CK polypeptide fraction (reaction with complex epithelia [CE] were useful differentiation markers for the four major groups of lung carcinomas. In one half of small cell carcinomas a positive reaction with NSE antibodies was found. S 100 and SC were good markers for papillary and bronchioloalveolar adenocarcinomas, whereas CEA was less important because of its reactivity with different types of lung carcinomas. To discern clear cell carcinomas of lung and renal origin a positive reaction with vimentin antibodies (some renal but not lung types) and with CA1 (no renal but all lung types) seemed to be useful. All hormone antibodies were of no importance as markers for difficult differential diagnosis, because positive reactivities were found in cases from every major carcinoma group. In addition, a Ca2+-activated adenosine triphosphatase (ATPase) was found in mesotheliomas but not in papillary adenocarcinomas.

Chromium content of normal and pathologically altered lung tissue.

Flameless atomic absorption spectrophotometry was used to determine the chromium (Cr) content of samples taken at autopsy from the lungs, bronchi, and regional hilar lymph nodes of 50 randomly selected patients from urban and rural areas; these patients were not known to have been excessively exposed to Cr.On the average, the Cr concentration in the lungs of patients younger than 40 yr of age was approximately 2 µg/g; for patients over 40, the average Cr values were between 5 and 15 µg/g dry wt. The highest values were found in samples from the apex of the lung. The Cr concentration in lung and lymph nodes increased in proportion to age and the degree of anthracosis. Chromium content in the bronchial wall was very low in all cases. Chromium values up to ten times greater as compared to age-matched average values were found in scarred lung tissue, probably caused by a postinflammatory lymph vessel blockade. Slightly elevated Cr values were found in smokers' lungs. Chromium values in tissue from primary lung carcinomas (n=9) were lower than those in neighboring lung tissue. Based on the results of this study the amount of Cr of lung and bronchial tissue does not appear to be associated with the induction of bronchial carcinoma.

In situ hybridization methods for the detection of somatostatin mRNA in tissue sections using antisense RNA probes.

In situ hybridization studies with [32P] and [3H] labelled antisense RNA probes were undertaken to determine optimal methods of tissue fixation, tissue sectioning, and conditions of hybridization, and to compare the relative merits of the two different radioactive labels. The distribution of somatostatin mRNA in neurons of rat brain using a labelled antisense somatostatin RNA probe was employed as a model for these studies. The highest degree of sensitivity for in situ hybridization was obtained using paraformaldehyde fixation and vibratome sectioning. Optimal autoradiographic localization of mRNA was obtained within 7 days using [32P] labelled probes. However, due to the high energy emittance of [32P], precise intracellular localization of hybridization sites was not possible. [3H] labelled RNA probes gave more precise cellular localization but required an average of 18-20 days autoradiographic exposure. The addition of the scintillator, PPO, decreased the exposure time for the localization of [3H] labelled probes to seven days. We also report a method for combined in situ hybridization and immunocytochemistry for the simultaneous localization of somatostatin in mRNA and peptide in individual neurons.

Immunocytochemical demonstration of intermediate filament cytoskeleton proteins in human endocrine tissues and (neuro-) endocrine tumours.

The presence and distribution of intermediate filament proteins, such as cytokeratins, vimentin, neurofilament proteins and glial fibrillary acidic protein were assessed immunohistochemically in pituitary adenomas, medullary thyroid carcinomas, endocrine pancreatic tumours, gastric, intestinal and bronchial carcinoids, parathyroid adenomas, pheochromocytomas, paragangliomas and related non-neoplastic tissues. In some cases, immunohistochemical results were correlated with cytoskeletal proteins as analysed by SDS-polyacrylamide gel electrophoresis. Cytokeratin antibodies with broad range of immunoreactivity (i.e. to murine liver cytokeratin component D) reacted with epithelial cells in all non-neoplastic endocrine tissues and related neuroendocrine tumours studied, except for adrenal medulla, pheochromocytoma and paraganglioma, independently of hormone production and biological behaviour. In contrast, antibodies to epidermis-derived cytokeratins failed to stain endocrine tissues and tumours. Paranuclear cytokeratin accumulations were seen in bronchial, gastric, and intestinal carcinoids and seem to be a common feature of neuroendocrine tumours. One- and two-dimensional SDS-polyacrylamide gel electrophoresis of non-neoplastic endocrine tissues and related tumours revealed two major keratin polypeptides corresponding to cytokeratins No. 8 and 18 of the cytokeratin catalog of human cells (Moll et al. 1982). According to this cytokeratin polypeptide composition, endocrine tissues and related tumours conform to the "simple type" of epithelia. Vimentin-related immunoreactivity was restricted to stromal cells and to folliculo-stellate cells in normal pituitary gland, Schwann cells in carcinoids and satellite cells in normal adrenal medulla and in pheochromocytomas. Neurofilament protein- (70 kD)-antibodies only stained nerve fibers in normal tissues and at the periphery of carcinoid tumour cell complexes, and to a variable degree, cells in nontumorous adrenal medulla, pheochromocytomas and paragangliomas. Furthermore, neurofilament reactivity was observed along with cytokeratin expression in two bronchial carcinoids.

Bronchial carcinoid arising in intralobar bronchopulmonary sequestration with vascular supply from the left gastric artery. Case report.

We report the unique case of a large, nonmetastasizing bronchial carcinoid tumor that arose within an intralobar bronchopulmonary sequestration in a 45-year-old man. The vascular supply to the sequestrated area within the left lower lobe as well as to the carcinoid tumor originated from atypical branches of the left gastric artery and the thoracic aorta. A left lower lobe lobectomy was performed. Histologically, a typical carcinoid tumor without lymph node metastases was found (T2 N0 M0). Seven years postoperatively, the patient is without signs of recurrence.

New immunocytochemical observations with diagnostic significance in cutaneous neuroendocrine carcinoma.

The presence and distribution of cytokeratins, actin, neurofilament protein, neuron-specific enolase, S-100 protein, and different neuropeptides were studied immunohistochemically by the peroxidase-antiperoxidase immunoenzyme method or the avidin-biotin-peroxidase technique in 10 patients with primary cutaneous neuroendocrine carcinoma. In all cases of cutaneous neuroendocrine carcinoma, immunoreactivity for neuron-specific enolase, cytokeratin, and neurofilament was identified. No staining was found after incubation with antibodies to S-100 protein, actin, and other tested neuropeptides. The cytoplasmic cytokeratin and neurofilament immunoreactivity was particularly strong in perinuclear areas, sometimes showing an annular pattern or displaying a discoid profile. The diagnosis of cutaneous neuroendocrine carcinoma may be reliably made by the immunocytochemical demonstration of neuron-specific enolase and intermediate filaments (cytokeratin, neurofilament protein) by conventional microscopy. Cutaneous neuroendocrine carcinoma has morphological, immunological, and histogenetic similarities to carcinoid neoplasms of the gut. We favor the concept that cutaneous neuroendocrine carcinoma is derived from, or differentiates toward, dermal neuroendocrine cells.

[The neuroendocrine Merkel cell carcinoma of the skin]. / Das neuroendokrine Merkelzellkarzinom der Haut.

Seven primary skin tumors from 5 women and 2 men were analyzed by light and electron microscopy and immunocytochemistry. The tumors were localized on the face (3 tumors) and on the extremities. The maximum diameter was between 1 and 3.5 cm. Two tumors metastasized to the regional lymph nodes 4 months after excision of the primary, 1 tumor metastasize to the regional lymph nodes after 5 years and the patient died of multiple metastases 8 years after excision of the primary on the forearm. No local recurrences developed. The tumors occurred in the dermis with frequent infiltration of the subcutaneous tissue. The epidermis was intact. The tumor cells formed large solid clusters, while their cytoplasm was faintly basophilic and formed a small rim round the large pale nucleus. In electron microscopy many cells displayed cytoplasmic electron-dense secretory granules with a mean diameter of approx. 100 nm. Immunocytochemistry showed that a large number of cells in all tumors contained neuron specific enolase and many cells of 4 tumors yielded formaldehyde-induced fluorescence. The tumors are therefore of neuroendocrine origin and may derive from merkel cells. They frequently give rise to erroneous diagnosis of metastasis of carcinoma or malignant lymphoma to the skin.