Evaluation of serum glycoprotein biomarker candidates for detection of esophageal adenocarcinoma and surveillance of Barrett’s esophagus

Esophageal adenocarcinoma (EAC) is thought to develop from asymptomatic Barrett’s esophagus (BE) with a low annual rate of conversion. Current endoscopy surveillance for BE patients is probably not cost-effective. Previously, we discovered serum glycoprotein biomarker candidates which could discriminate BE patients from EAC. Here, we aimed to validate candidate serum glycoprotein biomarkers in independent cohorts, and to develop a biomarker candidate panel for BE surveillance. Serum glycoprotein biomarker candidates were measured in 301 serum samples collected from Australia (4 states) and USA (1 clinic) using previously established lectin magnetic bead array (LeMBA) coupled multiple reaction monitoring mass spectrometry (MRM-MS) tier 3 assay. The area under receiver operating characteristic curve (AUROC) was calculated as a measure of discrimination, and multivariate recursive partitioning was used to formulate a multimarker panel for BE surveillance. Complement C9 (C9), gelsolin (GSN), serum paraoxonase/arylesterase 1 (PON1) and serum paraoxonase/lactonase 3 (PON3) were validated as diagnostic glycoprotein biomarkers in lectin pull-down samples for EAC across both cohorts. A panel of 10 serum glycoprotein biomarker candidates discriminated BE patients not requiring intervention [BE+/- low grade dysplasia] from those requiring intervention [BE with high grade dysplasia (BE-HGD) or EAC] with an AUROC value of 0.93. Tissue expression of C9 was found to be induced in BE, dysplastic BE and EAC. In longitudinal samples from subjects that have progressed towards EAC, levels of serum C9 were significantly (P\u3c0.05) increased with disease progression in EPHA (erythroagglutinin from Phaseolus vulgaris) and NPL (Narcissus pseudonarcissus lectin) pull-down samples. The results confirm alteration of complement pathway glycoproteins during BE-EAC pathogenesis. Further prospective clinical validation of the confirmed biomarker candidates in a large cohort is warranted, prior to development of a first-line BE surveillance blood test

Anatomical methods in cell death

This chapter describes methods for studying the morphology of cell death and the criteria used in identifying apoptosis and necrosis. Electron microscopy provides the most reliable method for recognizing the two processes; in many cases, however, they can be identified confidently using light microscopy alone. The recognition of apoptosis and necrosis is based primarily on the distinctive changes that take place within the affected cells. However, when these two processes occur in vivo, they also differ in their distribution and in the tissue reactions that are associated with them. These latter features may be of subsidiary use in identification. Thus, apoptosis involves scattered individual cells in a tissue, whereas necrosis involves groups of adjoining cells. Necrosis is accompanied by an acute inflammatory response with exudation of neutrophil leukocytes and monocytes; this event is characteristically absent in apoptosis. The light microscopic recognition of apoptosis depends on the detection of discrete well-preserved apoptotic bodies. Although convoluted budding cells are sometimes observed in smears, they are rarely seen in paraffin sections of immersion-fixed tissue

The role of apoptosis in the response of cells and tumours to mild hyperthermia

There is now abundant evidence that apoptosis, the cell death mechanism responsible for physiologial deletion of cells, can be triggered by mild hyperthermia. However, the overall importance of this mode of death in heated tumours has not yet been established. In this light and electron microscopic study, apoptosis induced by 43°C or 44°C water bath heating for 30 min in a range of murine and human tumours growing in vitro and in four murine tumours growing as solid nodules in vivo, was identified on the basis of its characteristic morphology, and the amount present quantified. Apoptosis was found to play a variable role in the response of tumours to heating, with the lowest levels produced in human melanoma lines (< 1%) and the highest levels in some Burkitt's lymphoma lines (up to 97%). In these latter tumours the induction of apoptosis is clearly a major component of the hyperthermic response

Ultrastructural localisation of epithelial mucin core proteins in colorectal tissue

Mucins are high molecular weight glycoproteins with a variety of postulated biological functions, including physicochemical protection from toxins and mutagens, adhesion modulation, signal transduction, and regulation of cell growth. Mucins are widely and differentially expressed in the gastrointestinal tract. To date, studies of cellular expression have relied on light microscopy using in situ hybridization and immunohistochemistry. Although informative, it has been difficult with these techniques to ascertain exactly which cell types are producing a given mucin. We studied expression of MUC1, MUC2, and MUC4 apomucins in a series of normal colon biopsies using a combination of immunoelectron microscopy and light microscopy. MUC1 mucin was localized to both goblet and columnar cells, where it was seen in secretory vesicles, microvilli, and in cytoplasmic remnants in goblet cell thecae. MUC2 expression was restricted to goblet cells, in which reactivity was concentrated in the rough endoplasmic reticulum (RER). MUC4 expression was seen in both columnar and goblet cells, localized to the RER. The inability to detect MUC2 and MUC4 apomucins in the Golgi complex and the mature mucous gel probably represents masking of peptide epitopes following O-glycosylation. This study has helped clarify lineage-specific mucin synthesis in the normal colon

Cell death induced by vincristine in the intestinal crypts of mice and in a human Burkitt's lymphoma cell line

Abstract. Although vincristine is widely used clinically in the treatment of some human cancers, its mechanism of action has not been clearly established. In this study, the patterns of cell death induced y vincristine in the intestinal crypts of mice and in a human Burkitt's lymphoma cell line were investigated by light and electron microscopy. Vincristine was found to enhance apoptosis of interphase cells in both systems and also to cause the arrest of cells in mitosis, the latter effect being ore pronounced in the intestinal crypts. Arrested mitotic cells went on to die by a process that had a number of features in common with apoptosis. These include compaction of chromatin (following coalescence of chromosomes), condensation of the cytoplasm, initial preservation of organelle integrity, and eventually the fragmentation of the cell into a number of membrane‐enclosed bodies which are morphologically similar to conventional apoptotic bodies. The results suggest that the cytocidal effect of vincristine is not solely dependent on metaphase arrest but is a cumulative one, resulting both from apoptosis of interphase cells and the ‘apoptotic‐like’ death of cells arrested in metaphase. Copyrigh

Increased cell surface protease activity in UV-irradiated cells undergoing apoptosis

UVB-irradiated HeLa cells undergoing apoptosis have increased cell surface protease (CSP) activity compared to viable or necrotic cells. In order to elucidate whether caspase 3 plays a role in the activation of CSP in cells undergoing apoptosis, HeLa cell cultures were pre-treated with the caspase inhibitor, DEVD, prior to being exposed to 500 Jm(-2) WE. DEVD significantly inhibited caspase 3 activity in cells undergoing apoptosis. but did not affect the activation of CSP in these cells. The findings suggest that the activation of CSP in apoptotic cells is unrelated to caspase 3 activity

Apoptosis in normal epithelium, premalignant and malignant lesions of the oropharynx and oral cavity: a preliminary study

To explore the involvement of apoptosis in the development of oral and oropharyngeal squamous cell carcinoma (SCC) in vivo, biopsies were taken from patients with macroscopically normal (n = 6), leukoplakic (n= 12) or malignant (n = 8) mucosa. Leukoplakic lesions were divided histologically into dysplasia (n=5) or carcinoma in situ (CIS: n=7). Material was prepared for light and electron microscopy. The apoptotic index (AI), vertical cell position of apoptoses (cp), mitotic index (MI) and AI:MI ratio were calculated for each patient. AI increased from 0.12% ± 0.07 S.E.M. (normal) to 0.58 ± 0.13 (CIS) but fell to 0.14 ± 0.14 in SCC. Apoptoses were suprabasal in normals, but generalised in CIS. MI increased from normal (0.20 ± 0.06) to SCC (0.32 ± 0.09), and AI:MI was at its maximum in CIS (1.57; SCC: 0.44). The results suggest that a change in apoptosis accompanies the onset of invasion in a premalignant lesion of the human oral cavity and oropharynx