Prognostic value of beta1,6-branched oligosaccharides in human colorectal carcinoma.

Increase of beta1,6-branched oligosaccharides is possibly associated with tumor progression and lymph node metastasis. The aim of this study was to determine the prognostic value of beta1,6 branches in human colorectal carcinoma. Expression of beta1,6 branches was histochemically evaluated using the leukoagglutinating Phaseolus vulgaris lectin, PHA-L, in 92 clinically documented colorectal carcinomas, of which 31 had formed lymph node metastases. The follow-up time ranged between 4 and 14 years (median, 10.3 years). A PHA-L staining index (SI), taking into account staining intensity and its percentage of tumor cut surface area, was established. The carcinoma SI was highly associated with the disease-free survival (P = 0.004) and overall survival (P = 0.005). Patients with a carcinoma SI of >1, as compared to those with a SI of < or =1, were at significantly higher risk for tumor recurrence, with a shorter disease-free survival (hazard ratio = 2.59, P = 0.005) and significant higher risk of death with shorter overall survival (hazard ratio = 2.51, P = 0.007). The carcinoma SI was also associated with the presence of lymph node metastases. We conclude that PHA-L staining in human colorectal carcinoma sections provides an independent prognostic indicator for tumor recurrence and patient survival and is associated with the presence of lymph node metastases.

RET proto-oncogene point mutations in sporadic neuroendocrine tumors.

We investigated the possible role of the RET proto-oncogene, which has recently been identified as the susceptibility gene for multiple endocrine neoplasia type 2, in the development of sporadic neuroendocrine tumors from different locations. DNA extracted from paraffin-embedded specimens of 112 neuroendocrine tumors was screened for somatic RET point mutations in exons 10, 11, 13, 15, and 16, where recently oncogenic mutations have been described in a subset of sporadic medullary thyroid carcinomas and pheochromocytomas. Methods employed included nonisotopic PCR-based single strand conformation polymorphism (PCR-SSCP) analysis, heteroduplex gel electrophoresis, and restriction enzyme digestion. The nucleotide sequence of samples with aberrant band patterns was identified by nonisotopic direct sequencing of PCR-amplified DNA. Forty-four percent (7/16) of sporadic medullary thyroid carcinomas and 15% (3/20) of pheochromocytomas contained a somatic, heterozygous point mutation at codon 918 of exon 16 (ATG --> ACG) causing a Met --> Thr substitution. None of the remaining 4 parathyroid adenomas, 8 pituitary adenomas, 17 pancreatic neuroendocrine tumors, 11 pulmonary and 10 gastrointestinal carcinoids, 7 small cell lung carcinomas, 5 neuroblastomas, 10 malignant melanomas, or 4 schwannomas contained mutations in any of the five RET exons tested. Although the numbers of each investigated neuroendocrine tumor type are small, our data indicate that oncogenic RET proto-oncogene mutations are involved in the formation of a subset of sporadically occurring medullary thyroid carcinomas and pheochromocytomas but do not appear to be generally important in the formation of other types of sporadically occurring neuroendocrine tumors.

Cystic fibrosis associated with neuronal intestinal dysplasia type B: a case report.

The authors present the case of a newborn girl who had cystic fibrosis associated with neuronal intestinal dysplasia type B (NID-B). The association is rare but must be considered in the differential diagnosis of gastrointestinal problems in patients with cystic fibrosis. The present case elucidates the intestinal problems that can arise with this combination of diseases. Although the unusual association found in this patient could have been a random occurrence, the possibility of an NID-B determining gene localized on chromosome 7q should be considered.

CD44 isoform expression in the diffuse neuroendocrine system. II. Benign and malignant tumors.

The membrane glycoprotein CD44 may be associated with aggressive behavior, dissemination, and poor prognosis of a variety of human tumors. In order to extend our knowledge on the expression and significance of CD44 in cells of the dispersed neuroendocrine system we investigated a spectrum of 134 neuroendocrine tumors, including pituitary adenomas, medullary thyroid carcinomas, parathyroid adenomas, pheochromocytomas, neuroblastomas, small-cell lung carcinomas, and bronchopulmonary, pancreatic, and gastrointestinal neuroendocrine tumors immunohistochemically for CD44 standard and variant exon-encoded gene products (CD44v3, -v4, -v5, -v6, -v9). Furthermore, we compared protein expression with that of CD44 mRNA by reverse-transcriptase PCR and Southern blot hybridization in a subset of tumors. Our results show that CD44 expression is correlated with the "histogenetic origin" of the appropriate neuroendocrine neoplasm. Endoderm-derived tumors generally express 3'-end CD44 variant exon-containing isoforms, whereas neural crest-derived tumors rarely are positive for CD44. Furthermore, we provide evidence that CD44 expression is not correlated with metastatic disease or a particular hormonal phenotype but exhibits an association with the degree of cellular differentiation. Thus, CD44 is not useful as marker for malignancy or prognosis. The number of patients with clinical follow-up data in our study was too small to allow definite conclusions about a possible correlation between CD44 expression and prognosis. But CD44 may help to better classify neoplasms with an unclear neuroendocrine phenotype.

CD44 isoform expression in the diffuse neuroendocrine system. I. Normal cells and hyperplasia.

Isoforms of the transmembrane glycoprotein CD44, which are generated by alternative splicing of nine variant exons, have been implicated in tumor cell adhesion, invasion and metastatic spread and may be indicators of the degree of tumor differentiation. Since little is known about the distribution of CD44 in non-neoplastic neuroendocrine cell types, we systematically investigated 42 samples of tissue from different organs, including the pituitary gland, thyroid, parathyroid, adrenal gland, lung, pancreas, stomach, duodenum, jejunum, ileum, appendix, and colon, immunohistochemically for the expression of CD44 standard and variant exon-encoded gene products (CD44v3, v4, v5, v6, v9). Furthermore, double immunolabeling for CD44 and a variety of peptide hormones was applied to characterize the different neuroendocrine cell types. Our results show that neuroendocrine cells derived from the neuroectoderm lack CD44 immunoreactivity. However, those originated from the endoderm exhibit a variable CD44 immunostaining which is related to their anatomical localization and the degree of differentiation irrespective of the hormone produced. Furthermore, we demonstrate that CD44 positive neuroendocrine cells predominantly express CD44 isoforms of the epithelial type and that hyperplastic clusters of neuroendocrine cells of pancreatic ducts express CD44 most probably as a sign of dedifferentiation.

Additive effects of interleukin 1 and tumour necrosis factor-alpha on the accumulation of the three granulocyte and macrophage colony-stimulating factor mRNAs in human endothelial cells.

The control of haematopoietic colony-stimulating factors (CSF) gene expression by interleukin 1 (IL-1) and tumour necrosis factor alpha (TNF-alpha) in cultured endothelial cells was studied by RNA hybridization and nuclear gene transcription. Both IL-1 and TNF-alpha induced, with somewhat different kinetics, a slow but marked accumulation of granulocyte-macrophage (GM)- and granulocyte (G)-CSF mRNAs in endothelial cells; macrophage (M)-CSF mRNA increased more rapidly but more moderately. Simultaneous treatment with maximally stimulating concentrations of both IL-1 and TNF-alpha had an additive effect on the accumulation of the three mRNAs, suggesting that both mediators act via independent pathways. The mechanism of CSF mRNA accumulation in endothelial cells was explored by nuclear run-on experiments, which showed that both IL-1 and TNF-alpha increase GM-CSF, G-CSF and M-CSF gene transcription to varying degrees.

[Expression of CD44 isoforms in normal human liver and also in regenerative and neoplastic liver changes]. / Expression von CD44 Isoformen in menschlicher normaler Leber sowie bei regenerativen und neoplastischen Leberveränderungen.

CD44 is a transmembrane glycoprotein of which a large number of isoforms exist. There is evidence, mostly from experimental systems, that isoforms of CD44 generated by alternative splicing of ten variant exons are involved in tumor invasion and metastasis formation. We have evaluated the expression of CD44 standard (CD44s) and variant exons (CD44v) encoded gene products in formalin-fixed and paraffin-embedded human liver specimens, using an immunohistochemical protocol with microwave-based antigen-retrieval and signal amplification. Tissue sections from normal, regenerative and neoplastic liver were studied. Our results indicate that: 1. in normal liver, both, hepatocytes and bile duct epithelia lacked detectable CD44s and CD44v containing isoforms; 2. most cirrhotic liver specimens were unreactive. Some regenerative nodules showed a focal weak positivity for CD44v5 and v9. Most proliferating bile ductules were weakly stained for CD44v9 and some of them also for v5 and v6; 3. most hepatocellular carcinomas displayed a heterogeneous staining of varying intensity for CD44v5, v6, v9 and CD44s. In a few tumors a weak staining for CD44v3 and v4 was also present. Furthermore, there was a tendency to an increased staining intensity of CD44 isoforms with decreasing differentiation; 4. cholangiocarcinomas showed a high expression of CD44s, v3, v5, v6 and v9 containing isoforms. We conclude that neoplastic transformation of hepatocytes and bile duct epithelia is associated with qualitative and quantitative changes in the expression of some CD44 variant exons encoded products. The clinical implications of these findings remain to be determined in a large series of patients.

Expression of CD44 isoforms and beta 1,6-branched oligosaccharides in human malignant melanoma is correlated with tumor progression but not with metastatic potential.

CD44, a family of closely related glycoproteins generated by alternative splicing, as well as the increased beta 1,6-branching of Asn-linked oligosaccharides (beta 1,6-branches), have been implicated in tumor progression and metastasis. We have investigated the expression of CD44 standard (CD44s), various CD44 splice variants (CD44v3, -v4, -v5, -v6 and -v9), and of beta 1,6-branches in a total of 37 paraffin-embedded human primary melanomas and metastases. Out of the 28 studied primary melanomas, 27 were positive for CD44s, 21 for CD44v5 (cytoplasmic staining) and 26 for beta 1,6 branches. Furthermore, superficial spreading melanomas showed a significant (p = 0.004) stronger staining for CD44s than the thick (> 1.5 mm) nodular melanomas, whereas no significant difference was found with regard to staining for CD44v5 and beta 1,6-branches. Eight of the 9 studied melanoma metastases were positive for CD44s, 6 for CD44v5 (cytoplasmic staining) and 7 for beta 1,6-branches. No CD44v3, -v4, -v6 and -v9 could be detected in any of the tumors. On average, metastases as compared to primary tumors, exhibited a significant (p = 0.002) weaker staining for CD44s. However, metastasizing melanomas could not be distinguished from non-metastasizing ones based on CD44 immunostaining.

CD44 standard and variant isoform expression in normal human skin appendages and epidermis.

CD44 isoforms have been implicated in tumor progression and metastasis formation. This study presents a thorough immunohistochemical analysis of CD44 standard and isoform expression in normal human skin appendages and epidermis applying monoclonal antibodies against CD44s, CD44v3, -v4, -v5, -v6, and -v9. An improved immunohistochemical protocol with microwave-based antigen retrieval in paraffin sections and heavy metal amplification of the diaminobenzidine reaction product provided enhanced resolution and sensitivity as compared to studies on frozen sections. The hair follicle, the seborrheic and eccrine sweat glands were strongly positive for all CD44 isoforms studied. In the latter, the clear cells but not the dark (intercalated) cells were positive. the sudoriferous ducts adjacent to the glands were weakly positive for all CD44 isoforms and strongly positive near the skin surface. In the apocrine glands, the basal cells showed only a moderate positivity. The myoepithelial cells expressed only CD44s. In the epidermis, all CD44 isoforms were detectable, with strongest CD44 immunostaining in the lower third of the stratum spinosum and weaker staining in the stratum basale and the upper two-thirds of the stratum granulosum. The stratum granulosum and corneum were unreactive. Thus, a regional and cell type-specific CD44 expression was revealed.

Colon carcinoma glycoproteins carrying alpha 2,6-linked sialic acid reactive with Sambucus nigra agglutinin are not constitutively expressed in normal human colon mucosa and are distinct from sialyl-Tn antigen.

In human colon carcinoma, increased amounts of sialic acids have been found and correlated with tumor progression. Further, the degree of O-acetylation of sialic acid residues in normal mucosa is higher than in colon carcinoma. Thus, tumor-associated sialylated antigens may be constitutively expressed in O-acetylated form in normal mucosa unreactive with the respective monoclonal antibodies. We have earlier demonstrated a colon carcinoma-associated expression of alpha 2,6-linked sialic acid residues with the Sambucus nigra agglutinin (SNA). We report now that de-acetylation of normal and transitional colonic mucosa, in contrast to sialyl-Tn antigen, does not result in SNA binding. Further, the alpha 2,6-linked sialic acid recognized by SNA is distinct from that of sialyl-Tn antigen. This is confirmed by Northern blotting detecting transcripts for alpha 2,6 sialyltransferase of N-glycoproteins and measurement of activity for this sialyltransferase. Blot analysis by SNA of colon carcinoma cells revealed few reactive glycoproteins. Quantitative differences in lectin labeling and sialyltransferase activity were found in HCT116 colon carcinoma cell sub-lines. Our data suggest that SNA binding in human colon carcinoma is due to de novo expression of a specific sialic acid present on selected glycoproteins.

CD44 standard and variant isoform expression in human epidermal skin tumors is not correlated with tumor aggressiveness but down-regulated during proliferation and tumor de-differentiation.

CD44 isoforms have been reported to be involved in tumor invasion and metastasis formation. Normal human skin expresses high levels of CD44 isoforms, but little is known about their expression in epidermal skin tumors. Expression of CD44 standard (CD44s) and variant exon (CD44v3, -v4, -v5, -v6, -v9)-encoded gene products has been studied in 74 benign, semi-malignant and malignant human epithelial skin tumors using a panel of well-characterized, variant exon-specific monoclonal antibodies (MAbs). Sensitivity and resolution of the immunohistochemical staining in paraffin sections was substantially improved by using microwave-based antigen retrieval and an optimized streptavidin-biotin-peroxidase technique. Immunostaining was evaluated semi-quantitatively and correlated with tumor type and degree of histological differentiation by non-parametric statistical tests. Furthermore, the relationship between CD44 expression and cellular proliferation rate as defined by the Ki-67 antigen was analyzed in basal cell carcinomas. We found a significant correlation between tumor type and CD44 isoform expression. Basal cell carcinomas exhibited the weakest staining and keratoacanthomas the strongest. Squamous cell carcinomas ranged in between, with a tendency to down-regulate CD44 expression upon de-differentiation. In basal cell carcinomas, an inverse relationship between CD44 expression and proliferation rate was directly demonstrated at the cellular level using double immunolabelling. Our data indicate that qualitative and quantitative changes in CD44 splicevariant expression in human skin tumors do not correlate with invasive and metastatic potential but are rather related to the degree of tumor differentiation.