Detection of inflammation- and neoplasia-associated alterations in human large intestine using plant/invertebrate lectins, galectin-1 and neoglycoproteins

Commonly, plant and invertebrate lectins are accepted glycohistochemical tools for the analysis of normal and altered structures of glycans in histology and pathology. Mammalian lectins and neoglycoproteins are recent additions to this panel for the detection of lectin-reactive carbohydrate epitopes and glycoligand-binding sites. The binding profiles of these three types of probes were comparatively analyzed in normal, inflamed and neoplastic large intestine. In normal colonic mucosa the intracellular distribution of glycoconjugates and carbohydrate ligand-binding sites in enterocytes reveals a differential binding of lectins with different specificity and of neoglycoproteins to the Golgi apparatus, the rough and smooth endoplasmic reticulum and the apical cell surface. The accessible glycoligand-binding sites and the lectin-reactive carbohydrate epitopes detected by galectin-1 show the same pattern of intracellular location excluding the apical cell surface. Lectin-reactive carbohydrate epitopes detected by plant lectins of identical monosaccharide specificity as the endogenous lectin {[}Ricinus communis agglutinin-I (RCA-I), Viscum album agglutinin (VAA)], however, clearly differ with respect to their intracellular distribution. Maturation-associated differences and heterogeneity in glycohistochemical properties of epithelial cells and non-epithelial cells (macrophages, dendritic cells, lymphocytes) are found. Dissimilarities in the fine structural Ligand recognition of lectins with nominal specificity to the same monosaccharide have been demonstrated for the galactoside-specific lectins RCA-I, VAA and galectin-1 as well as the N-acetylgalactosamine (GalNAc)-specific lectins Dolichos biflorus agglutinin (DBA), soybean agglutinin (SBA) and Helix pomatia agglutinin in normal mucosa and in acute appendicitis. Acute inflammation of the intestinal mucosa found in acute phlegmonous appendicitis is associated with selective changes of glycosylation of mucin in goblet cells mainly of lower and middle crypt segments resulting in an increase of DBA- and SEA-binding sites in the goblet cell population. Appendicitis causes no detectable alteration of neoglycoprotein binding. In contrast, tumorigenesis of colonic adenoma is characterized by increases in lectin-reactive galactose (Gal; Gal-beta 1,3-GalNAc), fucose and N-acetylglucosamine moieties and by enhanced presentation of respective carbohydrate ligand-binding capacity. This work reveals that endogenous lectins and neoglycoproteins are valuable glycohistochemical tools supplementing the well-known analytic capacities of plant lectins in the fields of gastrointestinal anatomy and gastroenteropathology

Cancer Biomarker Discovery: The Entropic Hallmark

Background: It is a commonly accepted belief that cancer cells modify their transcriptional state during the progression of the disease. We propose that the progression of cancer cells towards malignant phenotypes can be efficiently tracked using high-throughput technologies that follow the gradual changes observed in the gene expression profiles by employing Shannon's mathematical theory of communication. Methods based on Information Theory can then quantify the divergence of cancer cells' transcriptional profiles from those of normally appearing cells of the originating tissues. The relevance of the proposed methods can be evaluated using microarray datasets available in the public domain but the method is in principle applicable to other high-throughput methods. Methodology/Principal Findings: Using melanoma and prostate cancer datasets we illustrate how it is possible to employ Shannon Entropy and the Jensen-Shannon divergence to trace the transcriptional changes progression of the disease. We establish how the variations of these two measures correlate with established biomarkers of cancer progression. The Information Theory measures allow us to identify novel biomarkers for both progressive and relatively more sudden transcriptional changes leading to malignant phenotypes. At the same time, the methodology was able to validate a large number of genes and processes that seem to be implicated in the progression of melanoma and prostate cancer. Conclusions/Significance: We thus present a quantitative guiding rule, a new unifying hallmark of cancer: the cancer cell's transcriptome changes lead to measurable observed transitions of Normalized Shannon Entropy values (as measured by high-throughput technologies). At the same time, tumor cells increment their divergence from the normal tissue profile increasing their disorder via creation of states that we might not directly measure. This unifying hallmark allows, via the the Jensen-Shannon divergence, to identify the arrow of time of the processes from the gene expression profiles, and helps to map the phenotypical and molecular hallmarks of specific cancer subtypes. The deep mathematical basis of the approach allows us to suggest that this principle is, hopefully, of general applicability for other diseases

Analysis of the DNA content in the progression of recurrent and metastatic melanomas

Ploidy status and ploidy minted parameters of 18 primary melanomas, 32 recurrences and 18 lymphatic metastases were investigated applying CAS200 image analyzer All the tumours investigated were either suspicious for aneuploidy (Auer III) or clearly aneuploid (Auer IV). Primary melanomas differed from recurrent tumours concerning the percentage of aneuploid cells between 4c and 8c and 5c ER. Comparison of cutaneous tumors with lymphatic metastases showed a significant difference concerning the percentage of aneuploid cells between 2c and 4c. An already high aneuploidy rate in primary rumours suggests that recurrent and metastatic clones of cells are present in early stages and that aneuploidy status in primary melanomas could be regarded as one of the risk factors of recurrences and metastases

Detection of human papillomavirus DNA sequences in leucocytes: a new approach to identify hematological markers of HPV infection in patients with oral SCC

Squamous cell carcinoma of oral mucosa was shown to be associated with human papillomavirus (HPV) infection. The aim of this study was to find a hematological marker for HPV infection in patients with extensive HPV positive squamous cell carcinoma of the anterior oral cavity. In forty patients, referred to our clinic, suffering from histopathologically confirmed squamous cell carcinoma of the oral cavity with a tumor index T3/N2/M0 preoperative blood probes were taken. Human leucocytes were gained by FACS-lysis centrifugation. Paraffin-embedded sections of tumor tissue were deparaffinized and proteolytically digested to expose fixed target DNA.For detection of human papillomavirus, DNA sequences 6, 11, 16, 18, 31 and 33 a non radiocative in situ hybridization technique was used (Biohid®). For the first time we demonstrated HPV DNA sequences in leucocytes of patients with HPV positive oral SCC. Furthermore we found a correlation in type between HPV DNA sequences in tumor tissue and in leucocytes. This might lead to HPV as a hematological marker for follow up of patients with oral SCC and have consequences on therapy.Il a déjà été démontré que le cancer épidermoïde de la muqueuse orale pouvait être associé à une infection par le virus du papillome humain. Le but de cette étude a été de trouver un marqueur hématologique pour une infection à HPV chez des patients porteurs d’un cancer envahissant de la portion antérieure de la cavité orale HPV positif. Des prélèvements sanguins ont été effectués chez 4 patients adressés à notre clinique, présentant un cancer épidemoïde de la cavité orale histologiquement confirmé, avec un index tumoral T3/N2/M0. Les leucocytes humains ont été isolés par centrifugation FACSlyse. Des coupes incluses à la paraffine de tissus tumoraux ont été déparaffinés et soumises à une digestion par protéolyse afin d’être exposées à la fixation de la cible d’ADN.Une technique d’hybridation in situ non radio-acitve (Biohit®) a été utilisée afin de détecter les séquences 6, 11, 16, 18, 31 et 33 des virus du papillome humain. Nous sommes les premiers à avoir démontré la présence de séquences d’ADN de HPV dans les leucocytes de patients porteurs de cancers oraux HPV positifs. De plus, nous avons trouvé une corrélation entre les types des séquences d’ADN de HPV du tissu tumoral et des leucocytes. Il nous est possible ainsi de disposer d’un marqueur hématologique pour suivre l’évolution des patients porteurs de cancers de la bouche et d’envisager de nouvelles perspectives thérapeutiques

Lectin-binding sites in the epithelium of normal

By using histochemical methods, the binding pattern of various lectins in the epithelium of normal human appendix vermiformis was assessed. In addition to plant and invertebrate sugar receptors with nominal monosaccharide specificity for a-L-Fuc (UEA-1), a-DMan and a-D-Gluc (Con A), a-D-GalNAc (DBA), DGalNAc (SBA, HPA) B-D-Gal (RCA-I) and D-Gal (VAA), a mammalian B-galactoside-specific lectin (MW, 14 kDa) was included in the applied panel. The apical surface of enterocytes presented binding sites for RCA-1 on al1 cells, binding sites of UEA-1, DBA, SBA, HPA and VAA heterogeneously and no binding sites of Con A and 14 kDa. Binding sites of DBA, SBA, HPA, VAA and RCA-1 within enterocytes were located primarily focally in a supranuclear position, whereas Con A and 14 kDa bound to the cytoplasm both in apical and basa1 cell parts. In the follicle-associated epithelium more enterocytes expressed SBA- and VAA-binding sites than in the crypt epithelium. No differences between the lectin-binding pattern of M-cells and enterocytes were found in the follicle-associated epithelium. Intraepithelial macrophages were heterogeneously positive for the full panel of applied lectins. In contrast, intraepithelial lymphatic cells expressed binding sites only for RCA-1 and less prominently for Con A, VAA and 14 kDa. Goblet cell mucus contained lectin-binding sites in a heterogeneous manner: binding sites for Con A were not detected in goblet cells for DBA, SBA, VAA and 14 kDa in less than 20%, for UEA-1 in 20-40%, for HPA in 40-60% and for RCA-1 in 60-100% of the goblet cells. Secreted mucus differed in its lectin-binding capacity from intracellular goblet cell mucus selectively by an increase of UEA-1, SBA- and RCA-1-binding sites and a lack of 14 kDa-binding sites. Comparative study of lectin binding to goblet cell mucin in another region of the large intestine, namely the rectosigmoid, demonstrated that DBA, SBA and 14 kDa bound mainly to the dista1 colon, while UEA-1 and VAA labelling was selectively found in Oífprint reguests lo: Dr. Ulrich Brinck, M.D., Department of Pathology, University of Gottingen, Robert-Koch-Str. 40, D-37075 Gottingen, Germany apendiceal goblet cell mucin. Comparing the lectin-binding pattern in normal appendix epithelium and in appendicitis, the percentage of goblet cells expressing DBA- and SBA-binding sites in mucus globules was found to be about 4 times higher in appendicitis than in normal appendix. These results demonstrate that the expression of lectin-binding sites in appendiceal goblet mucin is specifically altered in appendicitis, indicating that there are selective changes of glycosylation of mucin in goblet cells mainly of the lower and middle crypt segment. Changes of lectinbinding pattern in appendicitis are discussed in connection with histochernical findings in inflammatory bowel disease

Analysis of the DNA mismatch repair proteins expression in malignant melanomas

The importance of properly functioning DNA mismatch repair has been shown in several tumour types both hereditary and sporadic but not yet in malignant melanomas. The aim of this study was to examine the expression of DNA mismatch repair genes (MLH1, MSH2, PMS1 and PMS2) in primary melanomas and to define their possible prognostic impact in 106 primary melanomas. MLH1 was found in 64 and MSH2 in 61 out of 106 melanomas. PMS1 and PMS2 proteins were present in 69 and 67 tumours, respectively. Loss of the expression of DNA mismatch repair proteins correlated with the increase of Clark levels. Cox regression analysis demonstrated some prognostic significance for PMS1 (forward p = 0.0018 and backward selections p=0.0277), MLH1 (only forward selection p =0.0081) and MSH2 (only backward selection p =0.0115)