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Fred T Bosman

Publications and source records attributed to Fred T Bosman.

At least 19 recordsLinked to original sources
PubMed2006

An agarose matrix facilitates sectioning of tissue microarray blocks.

Tissue microarray (TMA) is a powerful, high-throughput technique for in situ investigation of biomarkers in many tissue samples in a paraffin block by immunohistochemistry or fluorescence in situ hybridization (FISH), and has rapidly become the standard in marker studies. One of the difficult steps in the procedure is the sectioning of array blocks and mounting of sections using special slides and/or adhesive-coated tape, which demands specific experience and is time-consuming. We report an arraying method that allows melting of the receiving paraffin block and subsequent sectioning like any ordinary paraffin-embedded tissue block. The major difference from the standard microarray technique is the use of an agarose matrix in the recipient block. The agarose matrix allows melting of the paraffin without disturbing the array, resulting in perfect integration of the tissue cores. The agarose-paraffin TMA blocks limit tissue core loss during cutting, mounting, or immunohistochemical or FISH staining and better maintains the array.

ErbB Receptors↗
PubMed2006

Methylation of APC, TIMP3, and TERT: a new predictive marker to distinguish Barrett's oesophagus patients at risk for malignant transformation.

Barrett's associated oesophageal adenocarcinoma (EAC) is one of the most rapidly increasing malignancies in Western countries. Because of its poor prognosis, management of this disease through screening of Barrett's oesophagus (BE) patients and identification of those with a high risk of developing an adenocarcinoma seems a promising approach. Early molecular markers of malignant transformation might contribute to such screening approaches. Gene promoter methylation analysis was performed on normal, pre-neoplastic, and neoplastic lesions from BE patients. All lesions of interest were sampled by microdissection from formalin-fixed paraffin-embedded tissue sections. We found that, in 27 adenocarcinomas, APC, TIMP3, TERT, CDKN2A, and SFRP1 promoters were methylated in 93%, 65%, 64%, 48%, and 91%, respectively; in contrast MLH1, RASSF1, RARB, CDH1, and FHIT promoters were methylated in less than 5% of the tumours. In BE mucosa from patients who had progressed to adenocarcinoma (12 samples), APC, TIMP3, and TERT promoters were hypermethylated in 100%, 91%, and 92% of cases, whereas in BE mucosa from patients who had not progressed (16 samples) methylation was found only in 36%, 23%, and 17%, respectively. Furthermore, the epigenetic profile of BE with and without EAC differed significantly with, respectively, 81% and 26% of the PCR samples showing promoter hypermethylation for APC, TIMP3, and TERT (p < 0.0001). Promoter methylation of CDKN2A was infrequently detected in BE samples, while SFRP1 methylation was observed in all samples. Our results suggest that promoter methylation profiling of BE using multiple target genes including APC, TIMP3, and TERT might be used as a predictive marker for increased EAC risk.

Adenocarcinoma↗
PubMed2005

CTCF binds the proximal exonic region of hTERT and inhibits its transcription.

The expression of the catalytic subunit (hTERT) represents the limiting factor for telomerase activity. Previously, we detected a transcriptional repressor effect of the proximal exonic region (first two exons) of the hTERT gene. To better understand the mechanism involved and to identify a potential repressor, we further characterized this region. The addition of the hTERT proximal exonic region downstream of the hTERT minimal promoter strongly reduced promoter transcriptional activity in all cells tested (tumor, normal and immortalized). This exonic region also significantly inhibited the transcriptional activity of the CMV and CDKN2A promoters, regardless of the cell type. Therefore, the repressor effect of hTERT exonic region is neither cell nor promoter-dependent. However, the distance between the promoter and the exonic region can modulate this repressor effect, suggesting that nucleosome positioning plays a role in transcriptional repression. We showed by electrophoretic mobility shift assay that CCCTC-binding factor (CTCF) binds to the proximal exonic region of hTERT. Chromatin immunoprecipitaion assays confirmed the binding of CTCF to this region. CTCF is bound to hTERT in cells in which hTERT is not expressed, but not in telomerase-positive ones. Moreover, the transcriptional downregulation of CTCF by RNA interference derepressed hTERT gene expression in normal telomerase-negative cells. Our results suggest that CTCF participates in key cellular mechanisms underlying immortality by regulating hTERT gene expression.

Binding Sites↗
PubMed2005

[How important is the clinical context for a histopathological diagnosis?].

A histopathological examination consists of two distinct phases: observation and interpretation. As a rule, macro- and microscopical examination give strong diagnostic indicators and the diagnosis is made instantaneously: the two phases are strongly interwoven. Also, the clinical context often provides essential clues as to the final diagnosis. A request for a pathology examination must hence come with clinical informations and a specific question. Yet, the pathologist has to keep in mind that the interpretation of what he sees might follow too enthusiastically the proposed clinical diagnosis. It is hence preferable for the pathologist to take note of the clinical context only after the microscopic examination has been accomplished.

Diagnosis, Differential↗
PubMed2005

Immunohistochemical expression of endothelial markers CD31, CD34, von Willebrand factor, and Fli-1 in normal human tissues.

Few systematic studies have been published comparing the expression and distribution of endothelial cell (EC) markers in different vascular beds in normal human tissues. We investigated by immunohistochemistry the expression of CD31, CD34, von Willebrand factor (vWF), and Fli-1 in EC of the major organs and large vessels. Tissue samples obtained from autopsies and biopsy specimens were routinely processed and stained immunohistochemically for CD31, CD34, and vWF. Biopsy material was also stained immunohistochemically for Fli-1, D2-40, and Lyve-1. The expression pattern of the markers was heterogeneous in some of the organs studied. In the kidney, fenestrated endothelium of the glomeruli strongly expressed CD31 and CD34 but was only focally positive or completely negative for vWF. Alveolar wall capillaries of the lung strongly stained for CD31 and CD34 but were usually negative for vWF. The staining intensity for vWF increased gradually with the vessel caliber in the lung. Sinusoids of the spleen and liver were diffusely positive for CD31. They were negative for CD34 in the spleen and only expressed CD34 in the periportal area in the liver. Fli-1 was expressed in all types of EC but also in lymphocytes. D2-40 stained lymphatic endothelium only. Lyve-1 immunostaining was too variable to be applied to routinely processed tissues. The expression of EC markers CD31, CD34, and vWF in the vascular tree is heterogeneous with a specific pattern for individual vessel types and different anatomic compartments of the same organ. D2-40 labels lymphatic EC only.

Antigens, CD34↗
PubMed2004

Monoallelic methylation of the APC promoter is altered in normal gastric mucosa associated with neoplastic lesions.

Adenomatous polyposis coli (APC) promoter hypermethylation has been reported frequently in normal gastric mucosa, but it remained to be clarified whether this occurs in every individual. In this study, methylation of the APC promoter was analyzed in histologically normal-appearing gastric mucosa samples by methylation-sensitive single-strand conformation analysis and by a methylation-sensitive dot blot assay. Epithelial cell samples were collected by microdissection from tissue sections. Equal amounts of methylated and unmethylated APC alleles were found in all gastric mucosa samples from patients without any gastric lesions (20 samples). Allele-specific methylation analysis showed that the methylation of the APC promoter was monoallelic; however, which allele was methylated depended on the cell type. Increased or decreased methylation was found in 10 of 36 (28%) normal gastric mucosa samples adjacent to a gastric or esophageal adenocarcinoma. No allelic loss was found at the APC locus. Modification of the methylation status was also found in 3 of 21 (14%) normal-appearing gastric mucosa samples adjacent to intestinal metaplasia. In contrast, all normal mucosa samples in cases with chronic gastritis but without metaplasia or dysplasia showed a monoallelic methylation pattern. Our results indicate the following: (a) In normal gastric mucosa, the APC promoter shows monoallelic methylation, which is not due to imprinting but most likely due to allelic exclusion; (b) the excluded allele differs between foveolar and glandular epithelial cells; (c) the APC methylation pattern is frequently altered in normal-appearing gastric mucosa of gastric or esophageal adenocarcinoma patients; and (d) such alterations also occur in normal gastric mucosa adjacent to intestinal metaplasia.

Adenocarcinoma↗
PubMed2004

Immunohistochemical localization of hTERT protein in human tissues.

Telomerase is a ribonucleoprotein complex mainly composed of a reverse transcriptase catalytic subunit (telomerase reverse transcriptase gene, hTERT) that copies a template region of its RNA subunit to the end of the telomere. For detecting telomerase activity in a tissue specimen the TRAP assay is a relatively sensitive and specific method, but it can be used only on fresh tissue extracts and offers no information at the single cell level. Immunohistochemistry (IHC) allows to detect hTERT protein expression at an individual cell level in human tissues. We have tested commercially available anti-hTERT antibodies in formalin-fixed and paraffin-embedded human tissues by IHC. Only one monoclonal antibody (NCL-hTERT; Novacastra) was sufficiently specific and this was applied to human tissues in which telomerase activity had been shown by TRAP assay and hTERT mRNA expression by RT-PCR. hTERT protein localized diffusely in the nucleoplasm and more intensely in the nucleoli of cancer cells and proliferating normal cells. Mitotic cells showed diffuse staining of the entire cell. Granular cytoplasmic staining was occasionally found in some tumor cells. In telomerase-positive tumors not all the tumor cells showed hTERT immunoreactivity. A significantly heterogeneous hTERT protein expression was observed in human tumor tissues. The hTERT immunostaining in fixed tissues was concordant with telomerase activity and hTERT mRNA expression in corresponding non-fixed samples. Quantitative RT-PCR of microdissected sections showed that hTERT mRNA expression was higher in cells with nuclear expression than in those with cytoplasmic expression. Double staining with the M30 antibody showed that a subpopulation of hTERT-negative cells is apoptotic. We conclude that: (1) hTERT protein can be detected by IHC in fixed human tissues, but the choice of the antibody, tissue processing, and reaction conditions are critical, (2) hTERT protein localizes in the nucleoplasm, more strongly in the nucleolus, and occasionally in the cytoplasm, (3) telomerase-positive tumors show significant heterogeneity of hTERT protein expression, and (4) a subpopulation of hTERT protein negative tumor cells is identified as apoptotic cells.

Adenocarcinoma↗
PubMed2004

The cytoskeleton and disease.

Cytoskeletal research in recent years has revolutionized cell biology and biomedicine. The cytoskeleton spans the cytoplasm and interconnects the cell nucleus with the extracellular matrix, thereby forming a structural link between molecules involved in cell communication on the one hand, and gene expression on the other. Since the cytoskeleton is involved in virtually all cellular processes, abnormalities in this essential cellular component frequently result in disease. In this introduction, the basic structure of the cytoskeleton is briefly outlined. Furthermore, the disease processes in which the cytoskeleton plays a decisive role, and which are reviewed in detail in the papers in this issue, are briefly introduced. The advances in our understanding of the cytoskeleton and its function in disease will lead to new diagnostic and therapeutic applications in the foreseeable future.

Actins↗
PubMed2004

Academic medicine: dream or nightmare?

Academic medicine seems under pressure. The revolutionary changes in the practice of medicine of the last decades, the continuing demands for educational reform, decreased appreciation of the medical profession, budgetary constraints, and a variety of medical ethical issues have shaken up academic medicine and interest in an academic career seems to be waning. In this paper these issues are discussed from the viewpoint of a pathologist. The present situation can also be perceived as a challenge, which offers new opportunities. Better appreciation of educational efforts, more emphasis on the intellectual rather than the technological challenges of modern medicine, reconsideration of the strong tendency for super-specialization, recognition of originality and dedication over academic hierarchy and rethinking of strategies for development of patient-oriented research are briefly discussed.

Academic Medical Centers↗
PubMed2003

Implication of the exon region in the regulation of the human telomerase reverse transcriptase gene promoter.

The expression of the catalytic subunit (hTERT) represents the limiting factor for telomerase activity. In transfection studies, high level of activity of hTERT promoter is found, whereas low copy numbers of hTERT mRNA are detected in vivo. To explain this discrepancy, a series of vectors containing the hTERT promoter and gene were transiently transfected into HeLa cells. Four important regions were identified. First, the core promoter has bidirectional activity. Second, the distal upstream region (-1821 to -811bp) involved in the splicing of the first intron and could be a key of splicing specificity. Third, the intermediate promoter region (-800 to -300bp) could play an important role in silencing the reverse promoter activity. Fourth, the structural gene (up to +1077) strongly reduced hTERT promoter activity. These results provide the first evidence that the first two exons play a major role in the down-regulation of the hTERT promoter in telomerase-positive cells.

Codon, Initiator↗
PubMed2003

Functional structure and composition of the extracellular matrix.

In this brief introductory paper the general structure and the molecular composition of the extracellular matrix are outlined. Ultrastructural morphology of the extracellular matrix is introduced and subsequently the molecular structure of each of the main protein families, which together make up the extracellular matrix, is reviewed. Collagens, laminins, tenascins, and proteoglycans are addressed. An important common feature is the domain structure of these in general very large proteins. Several families have domains in common, which favours extensive interactions. Integrins play an important role in these interactions and also in the communication between cells and the matrix. The extracellular matrix appears to be a very dynamic structure, which has a prominent role in normal development as well as in a variety of disease processes. Matrix metalloproteinases are essential actors in this complex interplay between cells and the extracellular matrix.

Collagen↗
PubMed2003

Predictive factors in locally advanced rectal cancer treated with preoperative hyperfractionated and accelerated radiotherapy.

This study examines the prognostic significance of pathologic factors in patients with primary locally advanced rectal cancer treated prospectively with preoperative radiotherapy. From 1992 to 1998, 104 patients with rectal cancer of grades T3 or T4 and any N underwent preoperative radiotherapy followed by surgical resection. Survival curves were estimated according to the Kaplan-Meier method. Correlation of outcome with clinicopathologic variables (pathologic tumor and lymph node staging, histology, radial resection margin [RRM], clearance, vessel involvement, and tumor regression grade [TRG], quantitated in 5 grades) was evaluated using the Cox proportional hazards model. None of the patients achieved a histologically confirmed complete pathologic response, but 79% of the patients showed partial tumor regression (TRG2-4) and 21% did not show any tumor regression (TRG5). Among the tumors, 22% were of a mucinous type. The RRM was free of tumor in 76% of the surgical specimens. The median clearance was 2 mm. Vascular invasion was present in 37 cases (36%). In the univariate analysis, lymph node metastases, absence of tumor regression, positive RRM, and vascular invasion were correlated with adverse overall survival and disease-free survival; absence of tumor regression, positive RRM, and clearance <2 mm were correlated with local recurrences; and advanced pT stage was correlated only with disease-free survival. However, in the multivariate analysis, only lymph node metastases and RRM were independent prognostic factors for overall survival and disease-free survival, and clearance <2 mm was an independent prognostic factor for local control. Pathologic parameters remain strong determinants of local recurrence and survival in locally advanced rectal cancer, treated preoperatively with hyperfractionated and accelerated radiotherapy. We show that patients with advanced pT, positive lymph nodes, vascular invasion, positive RRM, clearance <2 mm, or absence of tumor regression are known to have poor clinical outcome.

Adult↗
PubMed2002

Expression of NDRG1, a differentiation-related gene, in human tissues.

NDRG1 is a member of the new N-myc downregulated gene (NDRG) family which belongs to the alpha/beta hydrolase superfamily, but without presenting a hydrolytic catalytic site. Diverse physiological and pathological conditions (hypoxia, cellular differentiation, heavy metal, N-myc, neoplasia) modulate NDRG1 transcription, mRNA stability, and translation. In this report we present the immunohistochemical localization of NDRG1 in a large set of normal human tissues at light and electron microscopic levels. The immunoreactivity of NDRG1 is mostly found in epithelial cells with different aspects. We observed NDRG1 primarily in the cytoplasm, but it is also associated with the cellular membrane and adherens junctions. Given the strong upregulation of NDRG1 under hypoxia and its nuclear localization, we propose a role for NDRG1 in protection from ischemic cell damage. The multiple localizations of this protein also suggest pleiotropic functions amongst which a functional involvement in the E-cadherin/catenin complex.

Adenocarcinoma↗
PubMed2002

Hypermethylation of the human telomerase catalytic subunit (hTERT) gene correlates with telomerase activity.

DNA methylation is an epigenetic process involved in embryonic development, differentiation and aging. It is 1 of the mechanisms resulting in gene silencing in carcinogenesis, especially in tumor suppressor genes (e.g., p16, Rb). Telomerase, the DNA polymerase adding TTAGGG repeats to the chromosome end, is involved in the regulation of the replicative life span by maintaining telomere length. This enzyme is activated in germ and stem cells, repressed in normal somatic cells and reactivated in a large majority of tumor cells. The promoter region of the hTERT gene, encoding for the catalytic subunit of human telomerase, has been located in a CpG island and may therefore be regulated at least in part by DNA methylation. We analyzed the methylation status of 27 CpG sites within the hTERT promoter core region by methylation-sensitive single-strand conformation analysis (MS-SSCA) and direct sequencing using bisulfite-modified DNA in 56 human tumor cell lines, as well as tumor and normal tissues from different organs. A positive correlation was observed among hypermethylation of the hTERT promoter, hTERT mRNA expression and telomerase activity (p < 0.00001). Furthermore, this correlation was confirmed in normal tissues where hypermethylation of the hTERT promoter was found exclusively in hTERT-expressing telomerase-positive samples and was absent in telomerase-negative samples (p < 0.00002). Since tumor tissues contain also nonneoplastic stromal elements, we performed microdissection to allow confirmation that the hTERT promoter methylation truly occurred in tumor cells. Our results suggest that methylation may be involved in the regulation of hTERT gene expression. To our knowledge, this is the first gene in which methylation of its promoter sequence has been found to be positively correlated with gene expression.

DNA Methylation↗
PubMed2002

Role of metallothioneins in irradiated human rectal carcinoma.

BACKGROUND: Metallothioneins (MT) are low-molecular weight, metal-binding proteins that play a role in cellular proliferation and differentiation, as well as in cellular defense mechanisms. They act as scavengers of free radicals produced by irradiation. A number of in vitro and in vivo studies have linked overexpression of cellular MT with tumor cell resistance to radiation. This is the first study that investigates whether MT expression is involved in the radioresistance of rectal carcinoma. METHODS: Using a mouse monoclonal antibody, MT expression was analyzed by immunohistochemistry on surgical samples (n = 85) from 85 patients with locally advanced rectal carcinoma who were treated preoperatively with a hyperfractionated and accelerated radiotherapy schedule and on tumor biopsies (n = 13) obtained before treatment. The potential correlations between MT expression and pathologic variables and survival were examined. RESULTS: MT were expressed strongly in both the cytoplasm and nucleus of tumor cells in 7 biopsy and 42 surgical samples. A comparison of MT expression in biopsy and surgical specimens showed that MT expression did not change after irradiation in most cases. Against all expectations, MT were expressed more frequently in tumors from responders than in those from the nonresponders (P = 0.02). There was no correlation between MT expression and tumor stage, histology after radiotherapy, or survival. CONCLUSION: These findings do not Cansupport the hypothesis that MT overexpression at the end of radiotherapy is a marker for radiation resistance.

Adult↗
PubMed2002

Importance of tumor regression assessment in predicting the outcome in patients with locally advanced rectal carcinoma who are treated with preoperative radiotherapy.

BACKGROUND: Locally advanced rectal carcinoma has a poor prognosis. However, since the introduction of preoperative radiotherapy, the outcome of patients with rectal carcinoma has been reported to have improved. Nevertheless, to the authors' knowledge few data are available regarding the histopathologic response to radiotherapy as assessed on surgical specimens as a potential predictive factor for outcome. METHODS: To estimate the effect of radiotherapy on rectal carcinoma, the authors retrospectively reviewed the surgical specimens of 102 patients with T3-4, N0 or > or = N1 rectal carcinoma and 1 patient with T2 but N1 rectal carcinoma. All patients were treated preoperatively with a hyperfractionated accelerated radiotherapy schedule in a prospective protocol (Trial 93-01). Using a standardized approach, tumor regression was graded using a system that varies from Grade 1 (tumor regression Grade [TRG] 1) when complete tumor regression is observed to Grade 5 (TRG5) when no tumor regression is observed. RESULTS: Radiotherapy resulted in tumor downstaging in 43% of the patients. There were 2 pT1 tumors (2%), 21 pT2 tumors (20%), 66 pT3 tumors (64%), and 14 pT4 tumors (14%) after treatment. Regional lymph nodes were involved in 55 patients (53%). None of the patients demonstrated a complete tumor regression after radiotherapy, but in 79% of the specimens a partial tumor regression was observed (TRG1: 0%; TRG2: 20%; TRG3: 39%; TRG4: 20%; and TRG5: 21%). The median actuarial overall survival (OS) and disease-free survival (DFS) were 52 months. Actuarial local recurrence rates at 2 years and 5 years were 6.4% and 7.6%, respectively. Univariate analysis showed the actuarial DFS to be significantly lower in patients with lymph node metastases (P = 0.0004) and advanced pT stages (pT3-4) (P = 0.03). A favorable outcome for OS, DFS, and local control was observed in patients with TRG2-4 (i.e., responders) compared with patients with TRG5 (i.e., nonresponders), but also in patients with low residual tumor cell density (TRG2, 3, and 4). On multivariate analysis, TRG remained an independent prognostic indicator for local tumor control. CONCLUSIONS: Tumor regression as well as residual tumor cell density were found to be predictive factors of survival in rectal carcinoma patients after preoperative radiotherapy. Even after preoperative radiotherapy, the pathologic stage of the surgical specimen remained a prognostic factor. The use of a standardized approach for pathologic evaluation must be implemented to allow comparison between the results of various treatment approaches.

Adult↗
PubMed2002

Application of molecular genetics to the diagnosis of lymphoid-rich effusions: study of 95 cases with concomitant immunophenotyping.

The cytological differentiation between reactive lymphocytosis and malignant lymphoma in serous effusions is often difficult. The present study was designed to evaluate the potential contribution of molecular genetic clonality analysis to a solution to this problem. We examined the cytological specimens of 95 consecutive patients collected during a 4-yr period, including 74 pleural, 20 peritoneal, and one pericardial fluids. Cytological diagnosis in the 95 lymphocyte-rich effusions was positive for lymphoma in 20 cases, suspicious for lymphoma in 26 cases, and negative in 49 cases. The analysis by ICC was not carried out, inconclusive, or noninterpretable in 25 cases. In five cases molecular genetic analysis was hampered by technical problems. By immunocytochemistry, eight additional cases of lymphoma were detected and lineage classification was achieved in 15 of the 20 cytologically positive effusions. PCR and Southern blot analysis were used to assess B- and T-cell clonality. Monoclonality was found in 40 (42%) of the 95 effusions analyzed. One-third of the effusions with a monoclonal B-cell gene rearrangement were detected by Southern blot analysis but not by the PCR performed in parallel. The results of molecular genetic analysis were corroborated by histological findings and/or clinical evolution in 15 cases. Our results indicate that molecular genetic analysis is a useful tool in the analysis of lymphocyte-rich serous effusions.

Adolescent↗