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Biomedical subjects

M Volkenandt

Publications and source records attributed to M Volkenandt.

At least 91 records · Page 5Linked to original sources
PubMed1993

Conformational polymorphisms of cRNA of T-cell-receptor genes as a clone-specific molecular marker for cutaneous lymphoma.

A novel molecular assay for the detection and characterization of monoclonal lymphoid populations in clinical specimens was developed. The assay is based on the principle that upon non-denaturing polyacrylamide gel electrophoresis RNA molecules separate into several metastable conformational forms. These conformational polymorphisms strictly depend on the nucleotide sequence of the individual molecule. Using DNA from formalin-fixed, paraffin-embedded tissue of patients with mycosis fungoides, highly variable junctional sequences of rearranged T-cell receptor gamma genes were amplified by polymerase chain reaction. Subsequently, the polymerase chain reactions products were transcribed into complementary RNA and analyzed by non-denaturing polyacrylamide gel electrophoresis. In clinical specimens with a monoclonal lymphoid population, a clone-specific pattern of bands was identified representing conformational polymorphisms of cRNA molecules of rearranged T-cell receptor gamma genes of the predominant lymphoid clone. Three biopsies from one patient taken from different sites of the body over 3 years yielded an identical pattern of bands. This methodology provides a novel and rapid tool for the molecular identification and characterization of clonal lymphoid populations in clinical specimens. It is likely to be of special value for studies on the clonal evolution of lymphoid disorders of the skin.

Base Sequence↗
PubMed1993

Molecular detection of clone-specific DNA in hypopigmented lesions of a patient with early evolving mycosis fungoides.

A specific molecular probe for the malignant lymphoid clone of lesions of a patient with mycosis fungoides was developed. As a clone-specific marker the junctional region of rearranged T-cell-receptor-gamma (TCR-gamma) genes was used. An oligonucleotide primer complementary to these sequences was designed. Using this primer and polymerase chain-reaction technology, early hypopigmented lesions of the patient, which were previously unclassifiable by conventional microscopy, were analysed. The study demonstrates, on the molecular level, that these hypopigmented lesions contain tumour clone-specific DNA and may represent early manifestations of mycosis fungoides.

Adult↗
PubMed1993

Lupus vulgaris confirmed by polymerase-chain reaction.

We report a case of lupus vulgaris with typical clinical and histological findings. Mycobacterium tuberculosis was not only identified by a conventional culture technique, but also by a recently established system which has been designed to detect mycobacterial DNA in formalin-fixed, paraffin-embedded tissue by polymerase-chain reaction (PCR). Because results can be obtained within days, the PCR-based technique may markedly facilitate the diagnosis of skin tuberculosis.

DNA, Bacterial↗
PubMed1993

Molecular detection of Borrelia burgdorferi in formalin-fixed, paraffin-embedded lesions of Lyme disease.

A system for the detection of a Borrelia burgdorferi (Bb)-specific gene segment in formalin-fixed, paraffin-embedded skin lesions is described. A nested polymerase chain reaction technique is used to selectively amplify in vitro a short segment of a Bb-specific gene recently described by Rosa et al. (J Infect Dis 1989: 160: 1018). The design of oligonucleotide primers for the amplification of a relatively short gene segment allows the successful analysis of DNA which has been altered by fixation in formalin. Using this technique, Bb-specific DNA was clearly identified in 8 of 12 specimens of erythema chronicum migrans and in 1 case of lymphadenosis benigna cutis. These skin lesions are known to represent cutaneous manifestations of Lyme disease. Negative control reactions, using DNA from borrelial strains not related to Lyme disease, were negative. The system enables the dermatopathologist to identify Bb in routinely fixed clinical specimens and allows the rapid analysis of various skin diseases for which an association with Bb so far has only been hypothesized.

Base Sequence↗
PubMed1993

Expansion of peripheral blood natural killer cells correlates with clinical outcome in cancer patients receiving recombinant subcutaneous interleukin-2 and interferon-alpha-2.

Natural killer (NK) cells are believed to contribute to the clinical efficacy of cancer immunotherapy using recombinant interleukin-2 (rIL-2) in humans. In previous trials of high-dose i.v. rIL-2, however, no correlation has been established between circulating NK cells and treatment response. Between January 1989 and October 1990, we treated a total of 47 outpatients with advanced tumors using low-dose s.c. rIL-2 and interferon-alpha-2 (rIFN-alpha). Therapy consisted of a 2-day rIL-2 pulse at 18 million IU/m2/day, followed by 6 weeks of rIL-2 (3.6 x 10(6)-4.8 x 10(6) IU/m2/day x 5 days/week) and rIFN-alpha (5 x 10(6)-6 x 10(6) U/m2 x 3/week). Before and after therapy, we phenotypically evaluated circulating lymphocytes and correlated them with clinical response. During 6-week therapy, peripheral blood lymphocytes bearing the CD56 (NK-cell-associated) surface antigen were increased significantly (p < or = 0.005) in treatment responders [complete response (CR) and partial response (PR), n = 10; 3.8-fold] and stable disease (SD) patients (n = 20; 2.1-fold), while patients with progressive disease (PD, n = 17) exhibited no significant expansion of circulating NK cells (p > 0.1). After one 6-week treatment cycle, CR/PR patients had significantly more peripheral NK cells, when compared with patients in SD (1.6-fold) and PD (1.9-fold) (p < 0.04). The overall number of circulating lymphocytes was also increased upon therapy (1.6-fold; p < or = 0.001), but remained independent of response (p > 0.4). These data demonstrate that s.c. rIL-2 and s.c. rIFN-alpha produce a significant increase in peripheral blood NK cells; this expansion correlates significantly with treatment response in advanced tumor patients receiving long-term combination immunotherapy at outpatient doses.

Antigens, CD↗
PubMed1993

Squamous cell carcinoma in localized scleroderma following immunosuppressive therapy with azathioprine.

A 40-year-old man presented with an ulcerated tumour in a fibrotic plaque on the dorsum of his left foot. Due to severe localized scleroderma, the patient had been treated with azathioprine 10 years earlier. Histopathology of the excised tumour revealed an anaplastic squamous cell carcinoma within a scar of localized scleroderma. The case demonstrates that not only patients with tense scar tissue following burning, congelation, chronic radiodermatitis, lupus vulgaris or lupus erythematosus but also patients who have had localized scleroderma may run a greater risk of developing squamous cell carcinoma. Immunosuppressive therapy has to be discussed as an additional risk factor in our patient. Therefore, narrow clinical follow-up was recommended for early detection of relapse.

Adult↗
PubMed1993

Mutations leading to antifolate resistance in Chinese hamster ovary cells after exposure to the alkylating agent ethylmethanesulfonate.

Chinese hamster ovary cells with a single allele for dihydrofolate reductase were used as a model system to study the effect of exposure to an alkylating agent, ethylmethanesulfonate, on rates and types of mutations at the dihydrofolate reductase locus leading to antifolate resistance. After overnight exposure to 400 micrograms/ml ethylmethanesulfonate, cells were allowed to recover for 3 days, and resistant colonies were selected in 8 x 10(-8) M trimetrexate. Trimetrexate, rather than methotrexate, was used as the selecting agent to increase the probability of obtaining mutations in dihydrofolate reductase, rather than in the reduced folate transport carrier protein. Seven of several hundred surviving colonies were selected at random, and cell lines were established. Cell lines 1-3 were maintained in culture in the presence of 8 x 10(-8) M trimetrexate and were 66-170-fold resistant to the drug. Cell lines 4-7 were initially expanded in 8 x 10(-8) M trimetrexate but were then maintained in the absence of the drug. These cell lines were 4.4-26-fold resistant to the drug, compared with the parental cell line. Cell line 1 was found to have an increase in dihydrofolate reductase activity, a corresponding increase in mRNA for dihydrofolate reductase, and amplification of this gene. Cell lines 2 and 6 had a mutated dihydrofolate reductase with altered trimetrexate- and methotrexate-binding properties. Cell line 3 had a 3-fold increase in dihydrofolate reductase activity. In cell lines 4, 5, and 7 the mechanisms of resistance to trimetrexate remain unknown.

Alkylating Agents↗
PubMed1992

Detection of point mutations in human DNA by analysis of RNA conformation polymorphism(s).

RNA molecules were found to separate into numerous metastable conformational forms upon non-denaturing gel electrophoresis. The equilibration of the conformations was accelerated by heating or mild denaturing conditions. Single-base substitutions in the sequence of the RNAs caused changes in the conformational patterns, including mobility shifts of major and minor conformations, appearance of new conformations and loss of other conformations. This sequence-dependent RNA conformational polymorphism was used to detect point mutations in p53 and, dihydrofolate reductase genes. Sense and anti-sense RNA strands corresponding to the same segment of the p53 gene gave entirely different conformational patterns. To generate the RNA, short regions of the target genes (up to about 250 bp) were amplified by the polymerase chain reaction and the resulting DNA segments transcribed to RNA by T7 RNA polymerase. The method is rapid, simple, amenable to non-radioactive visualization and was successful in several cases when DNA single-strand conformational polymorphism analysis (Orita et al. (1989) Genomics 5, 874-879) failed to detect the point mutation.

Base Sequence↗
PubMed1992

Quantitation of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase gene expression in human tumors using the polymerase chain reaction.

A polymerase chain reaction (PCR)-based method was used to quantitate the expression levels of low abundance genes relevant to cancer drug activity. RNA from tumor samples as small as 20 mg was isolated and converted to cDNA using random hexamers. The 5' primers for the PCR contained a T7 polymerase promoter sequence, allowing the PCR-amplified DNA to be transcribed to RNA fragments. In each sample, the linear ranges of amplification of each cDNA of interest were established. Relative gene expressions were calculated by extrapolating the amounts of PCR products generated within the linear amplification regions of each gene to equal volumes of the cDNA solution. The method was accurate to less than a 2-fold difference in expression levels. Using beta 2-microglobulin and beta-actin gene expressions as internal reference standards and cDNA from HT-29 cells as an external linearity standard, we measured the relative expressions of thymidylate synthase, dihydrofolate reductase, and DT-diaphorase in a number of clinical tumor samples. The expressions of these genes varied from 50- to 100-fold among different tumors, although most of the values were grouped within about a 10-fold range. The amount of thymidylate synthase gene expression in tumor tissues was directly proportional to the content of thymidylate synthase protein. Those tumors with the lowest thymidylate synthase expression had the best response to both the 5-fluorouracil-leucovorin and 5-fluorouracil-cisplatin combinations.

Actins↗
PubMed1992

p53 gene mutations in primary lung tumors are conserved in brain metastases.

The p53 product is frequently mutated in human tumors. Both acquired and inherited mutations have been described. These mutations transform p53 from a growth suppressor gene to a transforming oncogene. We examined tissue from 6 patients with primary lung carcinoma and the corresponding brain metastases for the presence of p53 mutations by immunohistochemistry. We then confirmed and characterized the mutations by single strand conformation analysis and by direct sequence analysis. All 6 patients had primary and metastatic tumor expressing a mutant p53. The mutations were all G-T transversions and mapped to exons 5, 6, 7, and 8. The mutations in the primary tumors were precisely conserved in the brain metastases.

Aged↗
PubMed1992

Analysis of the 14;18 translocation in cutaneous lymphomas using the polymerase chain reaction.

A series of 25 cutaneous B-cell lymphoid proliferations was analyzed for the presence of the (14; 18) translocation using the polymerase chain reaction. Junctional sequences of rearranged chromosomes 14 and 18 were amplified in vitro, and t (14; 18) specific sequences were detected in 1 of 14 primary cutaneous B-cell lymphomas, in 3 of 8 primary nodal B-cell lymphomas and in none of 3 B-cell pseudolymphomas. These results indicate that the t (14; 18) may occur in a small subset of primary cutaneous lymphoma. However, the difference in incidence of the t (14; 18) between primary nodal and primary cutaneous lymphomas suggests that different molecular mechanisms are involved in the pathogenesis of these lymphomas.

Base Sequence↗
PubMed1992

Sequence analysis of polymerase chain reaction amplified t(14;18) chromosomal breakpoints in formalin fixed, paraffin wax embedded follicular lymphoma.

AIMS: To determine whether junctional sequences of rearranged chromosomes can be amplified by use of the polymerase chain reaction (PCR) and whether direct sequence analysis of the PCR products is possible, using DNA from formalin fixed, paraffin wax embedded biopsy specimens. METHODS: DNA was extracted from paraffin wax embedded, formalin fixed lymphoma specimens, and junctional sequences of rearranged chromosomes were amplified by the PCR. The products were used as templates for asymmetrical PCR. Subsequently, direct sequence analysis was performed using the chain termination method. RESULTS: Formalin fixed, paraffin wax embedded biopsy specimens and PCR amplification could be used to determine the nucleotide sequences of junctional regions of rearranged chromosomes t(14;18) from patients with follicular lymphoma. CONCLUSION: The identification of junctional sequences of the translocation in follicular lymphoma provides a molecular "fingerprint" of t(14;18) of the lymphoma of an individual patient and can be used for the detection of clone specific DNA in any biopsy tissue obtained from the patient. The strategy used for rapid sequence analysis of PCR amplified DNA sequences will be useful in many areas of molecular pathology.

Base Sequence↗
PubMed1992

Overexpression of p53 protein in basal cell carcinomas of human skin.

Basal cell carcinoma (BCC) of the skin is the most common human cancer, but its molecular-genetic pathogenesis is unclear. In many other types of cancer, mutations of the tumor-suppressor gene p53 occur frequently and may lead to overexpression of a long-lived mutant form of p53 protein. In this study, overexpression of p53 protein was detected immunohistochemically in 30 (83%) of 36 specimens of BCC of the head and neck. The same regions of tumor typically were reactive both with a monoclonal antibody (PAb240) specific for the mutant protein and with one (PAb1801) directed against an epitope common to both wild-type and mutant p53 protein. Keratinocytes of chronically sun-exposed epidermis adjacent to BCCs also focally overexpressed p53 protein in the majority of cases, whereas those of sun-protected buttock skin did not. Mutation of p53 may form an important part of the pathogenetic sequence in a majority of cases of BCC.

Adult↗
PubMed1991

Development of a highly specific and sensitive molecular probe for detection of cutaneous lymphoma.

Using DNA from formalin-fixed paraffin-embedded tissue of a patient with mycosis fungoides, a highly specific and sensitive molecular probe for the malignant lymphoid cells of this patient was developed. Polymerase chain reaction (PCR) was used to selectively amplify the region of the rearrangement of TCR-gamma genes. Randomly inserted nucleotides between rearranged segments (N-region) were used as a specific marker for the malignant lymphoid clone of this patient. Clonal cells in paraffin-embedded tissue of previous and later biopsies were detected. This technique provides a new and unique tool for retrospective detection of early evolving cutaneous lymphoma, as well as for detection of minimal residual and systemic disease.

Aged↗
PubMed1991

Sequence analysis of DNA from formalin-fixed, paraffin-embedded human malignant melanoma.

Direct sequence analysis of in vitro amplified DNA from paraffin-embedded tissue is a promising new technique useful for detailed molecular analyses of material stored in dermatopathological archives. As only minute amounts of highly damaged DNA can be recovered from paraffin-embedded sections, sequence analysis of DNA from formalin-fixed and embedded tissue can result in less clear and readable data. We report here useful modifications that result in clear and readable sequence data. As an example, we detail the detection of a point mutation at codon 61 of the human N-ras gene in DNA from a formalin-fixed paraffin-embedded malignant melanoma specimen. This technique will be of value in many areas of molecular dermatopathology.

Base Sequence↗
PubMed1991

Mutational analysis of the human p53 gene in malignant melanoma.

Nine metastatic melanoma cell lines and two melanocyte cell lines were analyzed for point mutations in highly conserved regions of the p53 gene. No mutations were detected in the two melanocytic cell lines and in eight melanoma cell lines. However, a C----T transition at codon 248, resulting in a substitution of tryptophan for arginine, was found in one melanoma cell line. On immunohistochemical staining, only this cell line showed reactivity for mouse monoclonal antibody 1801, which is immunoreactive with human p53 protein. The original paraffin-embedded specimen from which this mutant cell line was established was obtained, and sequence analysis detected the identical mutation in the p53 gene as that seen in the derived cell line. This is the first report indicating point mutations in the p53 gene in malignant melanocytic tissues.

Base Sequence↗