Search PubMed⌕ Search

Biomedical subjects

H Kreipe

Publications and source records attributed to H Kreipe.

At least 37 records · Page 2Linked to original sources
PubMed2001

[Promoter hypermethylation during tumor progression: quantitative analysis employing kinetic PCR].

The methylation of cytosine residues in the promoter region of tumour-suppressor genes is now widely recognised as an alternative mechanism of gene inactivation in cancer. To address the question whether promoter methylation is an early event in the process of malignant transformation and whether quantitative changes are occurring during clonal evolution we developed a new assay for the measurement of cytosine methylation, which could also be applied to laser-microdissected archival specimens. We first improved the bisulfite conversion of genomic DNA in order to analyse cells isolated from archival tissue sections by laser-assisted microdissection. Analysing the methylation of key regulatory genes in archival specimens (n = 38) we could demonstrate that aberrant promoter hypermethylation is an early event during breast cancer development and changes quantitatively and in a gene-specific manner during progression. This novel methodological approach allows the morphology-guided quantitative analysis of changes in the methylation pattern during the clonal evolution of tumour cells. Quantitative differences detected by this assay will offer new insights into early steps in carcinogenesis and tumour cell heterogeneity.

DNA Methylation↗
PubMed2001

[Identification of new substrates of cyclin-activated kinases in breast cancers].

Mitogenic and growth-inhibitory signals influence cell-cycle progression through their action on a family of cyclin-dependent kinases (cdks). The activity of cdk complexes is regulated in part by the association of a cyclin partner that acts as a positive effector. These cyclin/CDK complexes promote cell cycle progression by the phosphorylation of key substrates. Cyclin D1 and E are frequently overexpressed in breast cancers and cyclin E overexpression has been correlated with a poor prognostic outcome. The in vivo substrates of cyclin E/CDK2 are, however, not well defined. We screened for cyclin E/CDK2 substrates in nuclear extracts of breast cancer cells as well as using a spotted-array protein library with purified active cyclin E/CDK2 complexes that were expressed in and purified from insect cells. Using this technique several potential cyclin E/CDK2 substrates were isolated. Further work is presently underway to identify these substrates and verify their authenticity as in vivo substrates of cyclin E/CDK2. These potential new substrates will help to unravel the highly complex mechanisms of cell cycle control and perhaps offer new targets for the diagnosis and/or treatment of breast cancer patients.

Breast Neoplasms↗
PubMed2000

Detection of gene amplification in archival breast cancer specimens by laser-assisted microdissection and quantitative real-time polymerase chain reaction.

Gene amplification is one of the most important mechanisms leading to deregulated gene expression in cancer. The exact quantitative detection of this frequent genomic alteration in solid tumors is often hampered by an admixture of nonneoplastic bystander and stroma cells. To overcome this obstacle and to develop an objective quantitative method we have combined laser-assisted microdissection of tumor cells with the novel 5'-exonuclease-based real-time polymerase chain reaction (PCR) assay. The latter method enables the highly reproducible exact quantification of minute amounts of nucleic acids. As a model system amplification of c-erbB2/Her-2/neu gene and the adjacent topoisomerase IIalpha gene was determined in paraffin-embedded breast cancer specimens (n = 23) after immunohistochemical labeling and laser-based microdissection of tumor cells. The high sensitivity of real-time PCR enabled the reliable and objective detection of low-level amplifications in as few as 50 cells from archival tissue sections. Low-level amplifications were shown to escape from detection unless tumor cells were isolated by microdissection. In selected cases intratumor heterogeneity was demonstrated using areas of approximately 50 to 100 cells. This novel approach combining immunohistochemistry, laser microdissection, and quantitative kinetic PCR allows morphology-guided studies in archival tissue specimens and will enable the exact quantification of gene copy numbers in even small and precancerous lesions.

Antigens, Neoplasm↗
PubMed2000

Polycythaemia vera: bone marrow histopathology under treatment with interferon, hydroxyurea and busulphan.

Little is known about long-term effects of myelosuppressive therapy on bone marrow of patients with polycythaemia vera, since histopathology from follow-up biopsies has not been frequently reported. Thus we conducted a retrospective morphometrical analysis of diagnostic and follow-up biopsies of 62 patients, evaluating fibre content, megakaryocytes and bone marrow cellularity. 8/62 patients were treated with interferon-alpha (INF), 11/62 with hydroxyurea (HU) and 11/62 with busulphan (BU). 32/62 served as controls; they were not treated with myelosuppressive drugs but with phlebotomy only. The median observation time was 2.3 yr. Results were compared on the basis of change per time. The bone marrow of the patients with phlebotomies only was characterised by increasing cellularity of haematopoesis, number and volume ratio of megakaryocytes and fibre content. In BU- and HU-treated patients, the haematopoesis was significantly reduced. The IFN patients revealed a reduction of cellularity which was not significant. The fibre content was reduced by BU only, but not significantly. No correlation between megakaryocytes and fibres was found. It could be concluded therefore that: 1) fibre proliferation within the bone marrow was not significantly altered by IFN, HU or BU. 2) Cellularity of haematopoesis was reduced significantly by HU and BU but only partly by IFN, corresponding with haematological remission.

Adult↗
PubMed2000

Common and epithelioid variants of hepatic angiomyolipoma exhibit clonal growth and share a distinctive immunophenotype.

Angiomyolipoma represents a rare liver tumor of uncertain histogenesis that is commonly considered a hamartoma. A series of 12 hepatic angiomyolipomas, including 3 samples of the epithelioid subtype, was analyzed for clonality using the human androgen receptor gene locus (HUMARA). Four of 6 informative cases revealed monoclonality. The polyclonal pattern in the 2 remaining cases was most probably caused by excessive infiltration of inflammatory cells. Monoclonality with an identical X-chromosomal inactivation pattern in all nodules was found in a multifocal recurrent tumor indicating a metastatic process. Despite the morphologic heterogeneity, all tumors displayed an identical immunohistochemical labeling pattern. It is concluded that different subtypes of hepatic angiomyolipoma exhibit a monoclonal and hence probably neoplastic growth and share an identical immunophenotype suitable for their identification even in small biopsy specimens. The epithelioid subtype may give rise to intrahepatic metastasis.

Adult↗
PubMed2000

Positive display of methylated sites: a novel method for the detection of promoter methylation.

Promoter methylation represents an important mechanism for silencing gene expression in higher eukaryotes. To study methylation of the promoter of the tumor suppressor p16INK4a, a fast and simple method was developed that, in contrast to previous studies, relies on the positive display of methylated sites (PDM). The method is based on bisulfite treatment of DNA, polymerase chain reaction (PCR)-amplification of the modified DNA and restriction digest of de novo created restriction sites to positively display DNA methylation in a background of unmethylated DNA. Since methylated as well as unmethylated DNA is amplified, information on the proportion of both is provided. Using this approach, 33 ductal invasive carcinomas, 4 normal mammary tissues, and 4 cell lines were analyzed for methylation. Methylation in the p16INK4a promoter was detected in 1 of 33 carcinomas (3%) and in 0 of 4 normal tissues. The conclusion is that PDM provides a useful tool in determining the degree and pattern of promoter methylation and is suitable to screen large series of tissue samples.

Base Pairing↗
PubMed2000

Detection of gene amplification in intraductal and infiltrating breast cancer by laser-assisted microdissection and quantitative real-time PCR.

Gene amplification is one essential mechanism leading to oncogene activation which is supposed to play a major role in the pathogenesis of invasive breast cancer. However, using standard methodologies the detection of gene amplifications has been limited especially in small-sized lesions, like pre-invasive precursor lesions. The combination of two novel technologies, laser-based microdissection and quantitative real-time PCR, facilitates the detection of low-level amplifications in morphologically defined lesions. As a model system we investigated in situ breast cancer (ductal carcinoma in situ, DCIS) classified according to the morphology-based Van Nuys grading system for amplification of growth-regulatory genes. In this study 83 formalin-fixed, paraffin-embedded archival DCIS specimens were examined after laser-based microdissection by quantitative real-time PCR using the TaqMan detection system for amplification of the c-erbB2, topoisomerase IIalpha, c-myc and cyclinD1 gene. In a subset of 17 DCIS with adjacent infiltrating tumour components we compared intraductal and invasive tumour components in parallel for differences in amplification status. The combination of these new techniques represents an excellent tool to gain new insights into carcinogenesis by analyzing genetic alterations in morphologically identified heterogeneous lesions in breast cancer progression within the very same specimen or even tissue slide.

Antigens, Neoplasm↗
PubMed2000

Laser-assisted microdissection and short tandem repeat PCR for the investigation of graft chimerism after solid organ transplantation.

The detection of donor-derived cells in the blood and tissues of graft recipients after solid organ transplantation is a readily observed phenomenon called microchimerism. Yet very little is known about the persistence and integration of recipient-derived cells in the transplanted organ, indicating a form of intragraft chimerism. To further study this phenomenon and its possible influence on graft acceptance or rejection, we developed the following novel approach. Immunohistochemically labeled cells were isolated by means of laser-based microdissection and subsequent laser pressure catapulting from paraffine-embedded posttransplantation biopsies. The following use of a highly sensitive PCR assay analyzing one polymorphic short tandem repeat (STR) marker enabled us to clearly identify the genotypes in samples containing as little as 10 isolated cells. The combination of laser-based microdissection and STR-PCR thus provides a powerful tool for the genotyping of even very few cells isolated from routinely processed biopsies after solid organ transplantation.

Cell Separation↗
PubMed2000

Quantitative molecular analysis of laser-microdissected paraffin-embedded human tissues.

Laser microdissection enables the contamination-free isolation of morphologically defined pure cell populations from archival formalin-fixed paraffin-embedded tissue specimens. Cells isolated by this method have been characterized by a wide variety of qualitative molecular assays, e.g. loss of heterozygosity, point mutations, clonality and lineage origin. The recently introduced real-time PCR technology renders the reliable quantification of very small amounts of nucleic acids possible. Several groups including our own showed that this technique can be successfully applied for the quantification of DNA and RNA isolated from microdissected archival tissue sections, even after immunohistochemical staining. The exact analysis of quantitative changes of nucleic acids during the course of pathological alterations has thus become possible. In many situations these quantitative changes can be expected to be more important than qualitative changes. The new technology for the quantification of structural genomic alterations and changes in the gene expression pattern in conjunction with microdissection have equipped morphologists with a powerful tool to study reactive and neoplastic changes of tissues.

Cell Separation↗
PubMed2000

Cytogenetic aberrations in primary and recurrent fibrolamellar hepatocellular carcinoma detected by comparative genomic hybridization.

Fibrolamellar hepatocellular carcinoma (FLC) is a rare entity of hepatocellular carcinoma (HCC) not yet analyzed cytogenetically. By using comparative genomic hybridization (CGH), we looked for chromosome changes in 2 primary FLCs and a recurrent FLC with and without metastases. CGH revealed an amplification of 1q in 1 primary FLC. The other primary FLC and a metastasis revealed no changes. The recurrent FLC showed 18 aberrations, including 1q+, 2p+, 3p+, 3q+, 4p+, 4q+, 5p+, 5q+, 6q+, 8p+, 8q+, 9q+, 12p+, 12q+, 18p+, 18q+, Xp+, and Xq+. In 2 metastases, 9 and 10 aberrations were seen, including 1q+, 3p-, 3q-, 4q+, 5p+, 5q+, 8q+, 10p+, 10q+, Xp+, and Xq+. In 9 cases of other entities of HCC, a mean of 10.2 aberrations per case were detectable affecting 1q (7 cases), 4q (5), 5q (4), 6q (5), 8p (5), 8q (5), 9p (4), 9q (5), 16q (4), 17p (5), and 17q (4). Chromosomes 2p, 2q, 3p, 3q, 4p, 5p, 6p, 7p, 7q, 10q, 11p, 11q, 12p, 12q, 13q, 14q, 16p, 18p, 18q, 20p, 20q, and 21q were altered in up to 3 samples. Our findings indicate striking differences in the number of chromosomal imbalances in primary FLC and recurrent FLC, whereas imbalances seen in the recurrent FLC and the other entities of HCC were similar in number and chromosomes involved. It may be speculated that these aberrations represent secondary events based on a genetic instability and do not mirror the primary alterations in these carcinomas.

Adult↗
PubMed1999

Primary intracerebral Hodgkin's disease: report of a case with Epstein-Barr virus association and review of the literature.

A case of primary intracerebral Hodgkin's disease (HD) without dural attachment in a 54-year-old immunocompetent patient is described. The infiltrate was located superficially in the occipital lobe and corresponded to the histologic type of nodular sclerosis. A typical immunohistochemical profile (membrane and cytoplasmic staining with dotlike Golgi enhancement of CD30, moderate cytoplasmic staining of CD15 in the Golgi area, membrane staining of CD20 of <10% of blastic cells, CD45RB negative) and in addition Epstein-Barr virus (EBV) latent membrane protein was detectable in Reed-Sternberg cells. Staging revealed no other organ sites of involvement. After combined surgery, postoperative radiotherapy, and chemotherapy, there are no signs of recurrence or systemic disease on follow-up for >1 year. To the authors' best knowledge, an association of EBV with primary central nervous system HD has not been demonstrated before.

Antineoplastic Combined Chemotherapy Protocols↗
PubMed1998

The cytomorphological spectrum of mantle cell lymphoma is reflected by distinct biological features.

Mantle cell (centrocytic) non-Hodgkin's lymphoma (MCL) is a malignant tumour with unique biological features. The pathogenesis of MCL seems to be strongly associated with aberrant function of the cell cycle. 110 cases of MCL have been analysed for their cytomorphological features, mitotic and proliferation indices, bcl-1 rearrangements, p53 expression patterns and DNA content by both interphase cytogenetic as well as DNA flow cytometric analyses. According to cytomorphology, three subtypes were recognized: a common, a lymphoblastoid and a pleomorphic variant of MCL. Blastic MCL subtypes were characterized by distinctly elevated mitotic and proliferation indices, frequent bcl-1 rearrangements at the MTC locus, and overexpression of p53. The most interesting finding, however, was a striking tendency of blastoid MCL subtypes to harbour chromosome numbers in the tetraploid range, a feature clearly separating these neoplasms from other types of B-cell NHL and possibly being related to its unphysiological expression of cyclin D1. Although characterised by a uniform immunophenotype and common biological background, MCL shows a broad spectrum of morphological features ranging from small cell to blastic types, and this spectrum is mirrored by distinct biological features.

Cell Division↗
PubMed1997

Cyclin D1 expression in mantle cell lymphoma is accompanied by downregulation of cyclin D3 and is not related to the proliferative activity.

The cell cycle regulatory protein cyclin D1 is essential for G1-S phase transition in several epithelial and mesenchymal tissues but is apparently not essential in normal mature B cells. An overexpression of cyclin D1 is induced by the chromosomal translocation t(11;14)(q13;q32), which characterizes non-Hodgkin's lymphomas (NHLs) of mantle cell type. We studied 26 cases of mantle cell lymphoma (MCL) for the expression of cyclins D1 and D3. A total of 23 lymphomas showed a nuclear staining for cyclin D1, whereas reactive B cells of residual germinal centers were constantly negative. When compared with cyclin D3, an inverse staining pattern emerged. Whereas the B cells of residual germinal centers reacted strongly positive for cyclin D3, there was low or missing expression of cyclin D3 in MCL cells. In other B-cell lymphomas (n = 55), including chronic lymphocytic leukemia, low-grade lymphomas of mucosa-associated lymphatic tissue, follicular lymphomas, and diffuse large B-cell lymphomas, no cyclin D1 expression could be detected and 89% of these cases displayed cyclin D3 positivity. Lymphoma cell lines harboring the t(11;14) showed cyclin D1 protein but no or very low levels of cyclin D3; three other B-cell lines, a T-cell line, and peripheral blood lymphocytes strongly expressed cyclin D3 and reacted negatively for cyclin D1. We conclude that the chromosomal translocation t(11;14) leads to an abnormal protein expression of cyclin D1 in the tumor cells of MCL and induces a consecutive downregulation of cyclin D3. In contrast to other B-NHLs, cyclin D1 and D3 expression in MCL is not related to the growth fraction.

Cell Division↗
PubMed1997

Cell cycle-coupled relocation of types I and II topoisomerases and modulation of catalytic enzyme activities.

We visualized DNA topoisomerases in A431 cells and isolated chromosomes by isoenzyme-selective immunofluorescence microscopy. In interphase, topoisomerase I mainly had a homogeneous nuclear distribution. 10-15% of the cells exhibited granular patterns, 30% showed bright intranucleolar patches. Topoisomerase II isoenzymes showed spotted (alpha) or reticular (beta) nuclear patterns throughout interphase. In contrast to topoisomerase IIalpha, topoisomerase IIbeta was completely excluded from nucleoli. In mitosis, topoisomerase IIbeta diffused completely into the cytosol, whereas topoisomerases I and IIalpha remained chromosome bound. Chromosomal staining of topoisomerase I was homogeneous, whereas topoisomerase IIalpha accumulated in the long axes of the chromosome arms and in the centriols. Topoisomerase antigens were 2-3-fold higher in mitosis than in interphase, but specific activities of topoisomerase I and II were reduced 5- and 2.4-fold, respectively. These changes were associated with mitotic enzyme hyperphosphorylation. In interphase, topoisomerases could be completely linked to DNA by etoposide or camptothecin, whereas in mitosis, 50% of topoisomerase IIalpha escaped poisoning. Refractoriness to etoposide could be assigned to the salt-stable scaffold fraction of topoisomerase IIalpha, which increased from <2% in G1 phase to 48% in mitosis. Topoisomerases I and IIbeta remained completely extractable throughout the cell cycle. In summary, expression of topoisomerases increases towards mitosis, but specific activities decrease. Topoisomerase IIbeta is released from the heterochromatin, whereas topoisomerase I and IIalpha remain chromosome bound. Scaffold-associated topoisomerase IIalpha appears not to be involved in catalytic DNA turnover, though it may play a role in the replicational cycle of centriols, where it accumulates during M phase.

Antibody Specificity↗
PubMed1997

Invasion and destruction of mucosal plasma cells by Tropheryma whippelii.

Whipple's disease is a poorly understood systemic disorder associated with the bacillus, Tropheryma whippelii. An early stage of Whipple's disease is studied by using electron microscopy (ELMI) and immunohistochemistry. The diagnosis was confirmed by polymerase chain reaction-amplification and sequencing of the 16S ribosomal-RNA of the bacterium. By using ELMI, Tropheryma whippelii was found in plasma cells and macrophages in the jejunal mucosa. The immunoglobulin (Ig)A-positive plasma cells were focally destructed and their number significantly reduced. However, the bacilli in the plasma cells were morphologically intact. In contrast, the macrophages showed no signs of cell destruction, but contained bacilli in various stages of disintegration. A cytopathic effect of Tropheryma whippelii to IgA plasma cells may be the reason for the commonly found plasma cell reduction in the small intestine mucosa and an important pathogenic mechanism contributing to the evasion of the bacilli from local immune response.

Actinobacteria↗
PubMed1997

[Clonal analysis in cells using PCR and laser microdissection].

Clonality represents one of the hallmarks of neoplastic cell growth. X-chromosomal inactivation patterns have been used to determine clonality in various tumors. This approach is limited by admixture of polyclonal non-tumor stroma cells among which the monoclonal proliferation may be missed. In order to overcome this limitation, we combined a sensitive PCR based DNA analysis with a highly selective microdissection technique using a laser beam. In sections of intraductal mammary carcinomas tumor cell complexes of at least 100 cells were isolated by removing the surrounding stroma by laser irradiation. Thereby, tumor cells could be isolated without contaminating non-neoplastic elements. Clonality in these cells was determined using two X-chromosomal polymorphic sites-phosphoglycerate kinase 1 (PGK1) and human androgen receptor (HUMARA). Control experiments could show the polyclonal nature of the surrounding tissue. Moreover, complete destruction of DNA by laser irradiation was assured. The technique requires a certain amount of cells and DNA in order to avoid artefacts that result from preferential amplification of exclusively one X-chromosomal allele in small samples. We conclude that combination of laser-microdissection with PCR analysis of X-chromosomal inactivation patterns enables the detection of clonal cell populations in heterogeneous tissues. Studies of clonality in borderline cases between reactive and neoplastic proliferations or premalignant lesions are made possible by this technique.

Breast Neoplasms↗