RESUMEN
Hereditary Non-polyposis Colorectal Cancer (HNPCC) is an autosomal dominant cancer predisposition syndrome caused by germline mutations in at least four genes encoding integral components of the cellular DNA mismatch repair (MMR) system. The spectrum of genetic alterations encompasses missense- and nonsense mutations, intronic mutations affecting splice donor or acceptor sites as well as small-scale deletions and insertions. We have identified a 'nonsense' mutation that activates a cryptic splice site generating an in frame deletion of the last 17 codons of exon1 of the hMLH1 gene causing HNPCC in a German family. We present a comprehensive genetic analysis of this family that demonstrates important aspects of HNPCC pathogenesis.
Asunto(s)
Proteínas Portadoras/genética , Codón sin Sentido , Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Proteínas Nucleares/genética , Empalme del ARN , Proteínas Adaptadoras Transductoras de Señales , Adulto , Humanos , Masculino , Homólogo 1 de la Proteína MutLRESUMEN
The aim of the study was to explore distress and health beliefs before and after comprehensive interdisciplinary counseling in families at risk for hereditary non-polyposis colorectal cancer (HNPCC). Results reported here were derived from a consecutive sample of 65 counselees [31 patients with colorectal cancer (CRC) and 34 unaffected at-risk persons] who participated in interdisciplinary counseling provided by human geneticists, surgeons, and psycho-oncologists before genetic testing. Data were collected from self-administered questionnaires before, as well as 4-6 weeks after, counseling. Distress and perceptions specific to HNPCC were assessed at both timepoints using standardized as well as author-derived instruments. Distress declined after counseling, as did worries related to HNPCC. An increase was found in personal belief in control of cancer risk, for instance, in the perceived efficacy of early detection of CRC. We also observed a trend toward greater anticipated ability to cope with a positive gene test after counseling. Changes after counseling were generally more pronounced for persons at risk, as compared to patients with cancer. The decrease in distress was partly attributable to an increase in personal self-confidence. One-third of the sample reported enhanced communication specific to hereditary disease within the family after counseling. A substantial minority, however, said they experienced increased worry and physical symptoms after counseling. Overall, counselees demonstrated less stress and perceived cancer threat as well as enhanced beliefs regarding personal control over cancer, suggesting an overall beneficial impact of comprehensive counseling. Further research is needed to identify those individuals most at risk for increased fear and worry related to HNPCC so that they may be most appropriately counseled.
Asunto(s)
Neoplasias Colorrectales Hereditarias sin Poliposis/genética , Asesoramiento Genético/psicología , Predisposición Genética a la Enfermedad , Adulto , Anciano , Femenino , Predisposición Genética a la Enfermedad/psicología , Humanos , Masculino , Persona de Mediana EdadRESUMEN
A cohort of 70 consecutive women at a university hospital colposcopy clinic with untreated CIN I and CIN II (CIN I/II) confirmed by cytology and histology was followed for 1 year in the setting of a prospective trial. In the lesions, the presence of DNA from HPV types was examined by restriction fragment length polymorphism (RFLP) analysis. Aneuploid cell lines were demonstrated by aneuploid histograms generated by high-resolution DNA flow cytometry. HPV type 16 infection and the existence of aneuploid cell lines proved to be significant risk factors for CIN I/II lesions to persist or progress to CIN III in the 1-year follow-up period in the same cohort of patients. The relative risks and 95% confidence intervals (CI) were 1.81 (1.44-2.76) for aneuploid cell lines and 1.74 (1.10-2.76) for HPV type 16 infection in CIN I/II lesions. As a predictive diagnostic test for CIN I/II lesions to persist or progress, the specificity and positive predictive value (PPV) for aneuploid histograms were 100% (CI, 73.5-100%) and 100% (CI, 86.8-100%), respectively. The low sensitivity of 27.3% (CI, 14.9-42.8%) restricted the clinical application of the test, leaving 32 of 44 women with persisting or progressing CINI/II with diploid histograms. HPV type 16 positivity by FRLP had a PPV of 68.4% (CI, 43.5-87.4%) as a prognostic test. Six of 19 HPV 16 infected women showed complete remission of their CIN lesion. A combination of the two tests did not provide any additional information.
Asunto(s)
Citometría de Flujo/métodos , Papillomaviridae/genética , Displasia del Cuello del Útero/diagnóstico , Displasia del Cuello del Útero/virología , Adolescente , Adulto , Aneuploidia , Línea Celular , Estudios de Cohortes , Progresión de la Enfermedad , Femenino , Estudios de Seguimiento , Humanos , Persona de Mediana Edad , Polimorfismo de Longitud del Fragmento de Restricción , Estudios Prospectivos , Factores de Riesgo , Displasia del Cuello del Útero/genéticaRESUMEN
PURPOSE: Cytokeratin 20 (CK 20) is selectively expressed in urothelium, gastric intestinal epithelium, in Merkel cells and in a variety of malignant neoplasms. CK 20 RT-PCR assay has been extensively used to detect isolated cancer cells in peripheral blood, lymph nodes and bone marrow samples of patients with colorectal carcinoma. Since CK-20 is also actively expressed in transitional cell carcinoma (TCC), we analyzed, whether CK 20 Reverse Transcriptase Polymerase Chain Reaction (RT-PCR) is suitable to detect residual tumor cells in patients with transitional cell carcinoma of the bladder and the upper urinary tract. MATERIALS AND METHODS: Nested Reverse Transcriptase Polymerase Chain Reaction assay was used to analyze CK 20 transcripts in peripheral venous blood samples and tumor biopsies of 49 patients with transitional cell carcinoma. Blood samples of 22 healthy volunteers served as negative controls. RESULTS: CK 20 mRNA was detectable in blood samples of 12 of 49 patients with TCC. All blood samples of the control group tested negative. The detection rate for CK 20 mRNA significantly correlated (p = 0.0019, Cochran-Armitage Trend Test) to the stage of disease and increased from 0% in stage pTa to 63% in stage pT4. CONCLUSIONS: These results suggest that CK 20 is a suitable marker for the detection of disseminated TCC cells in peripheral venous blood samples and may be helpful in the molecular staging of TCC patients. The prognostic relevance has to be evaluated in further followup.
Asunto(s)
Biomarcadores de Tumor/análisis , Carcinoma de Células Transicionales/patología , Proteínas de Filamentos Intermediarios/análisis , Células Neoplásicas Circulantes/patología , Neoplasias de la Vejiga Urinaria/patología , Anciano , Biomarcadores de Tumor/genética , Carcinoma de Células Transicionales/química , Femenino , Humanos , Proteínas de Filamentos Intermediarios/genética , Queratina-20 , Masculino , Estadificación de Neoplasias , Células Neoplásicas Circulantes/química , ARN Mensajero/análisis , Reacción en Cadena de la Polimerasa de Transcriptasa Inversa , Neoplasias de la Vejiga Urinaria/química , Neoplasias Urológicas/química , Neoplasias Urológicas/patologíaRESUMEN
Colorectal cancer is considered a non-immunogenic malignany. One strategy to augment the immunogenicity of such tumours is represented by the expression of costimulatory molecules by gene transfer. Using transfected variants of the human colorectal cancer cell line SW480 we tested various costimulatory molecules (CD80, CD86, CD54) and a class II major histocompatibility complex (MHC) allele (HLA-DR3) alone or in combination on their ability to support primary T-lymphocyte activation in vitro. Expression of CD80 or CD86 similarly as the combination of both was not sufficient to induce proliferation of human allogeneic T cells. Expression of CD54 together with CD80 strongly augmented the costimulatory function of CD80, as observed in the presence of a CD3 monoclonal antibody (mAb), but did not lead directly to a T-cell response against modified tumour cells. Importantly, SW480 cells coexpressing CD54, CD80 and the HLA-DR3 allele effectively promoted T-lymphocyte proliferation. Moreover, the use of such CD54+/CD80+/HLA-DR3+ SW480 variants for repetitive stimulations resulted in the generation of T-cell lines predominantly composed of CD8+ T cells exhibiting class I MHC restricted cytolytic activity towards untransfected SW480 tumour cells. This demonstrates that the generation of immunogenic tumour cell variants, i.e. for the use as cellular vaccines, requires multiple genetic alterations in the case of non-immunogenic human tumours cells, such as colorectal cancer cells.
Asunto(s)
Antígeno B7-1/genética , Neoplasias Colorrectales/inmunología , Técnicas de Transferencia de Gen , Antígeno HLA-DR3/genética , Molécula 1 de Adhesión Intercelular/genética , Activación de Linfocitos , Linfocitos T CD8-positivos/inmunología , División Celular , Expresión Génica , HumanosRESUMEN
Bacterial cytosine deaminase (CD) converts the non-toxic prodrug 5-fluorocytosine (5-FC) into 5-fluorouracil (5-FU), which is toxic for mammalian cells. Therefore, the CD gene is used in cancer gene therapy to achieve high local concentration of a toxic metabolite without significant systemic toxicity. To allow the detection of CD expression at the protein level, we raised both polyclonal rabbit antisera and a monoclonal antibody (mAb) against a histidine-tagged CD fusion protein. The specificity of the polyclonal antisera and the mAb was confirmed by immunohistochemistry, immunoblot analysis, and immunoprecipitation using CD-expressing tumor cell lines. Furthermore, the antibodies can be used for ELISA assays and flow cytometry. Finally, the CD protein could be demonstrated in frozen tissue sections of CD-modified tumors in a rat tumor model using the anti-CD serum. With these antibodies, CD expression can now be monitored throughout in vitro and in vivo gene transfer studies, including clinical protocols relying on the CD suicide gene strategy.
Asunto(s)
Anticuerpos Monoclonales , Especificidad de Anticuerpos , Nucleósido Desaminasas/análisis , Animales , Western Blotting , Citosina Desaminasa , Ensayo de Inmunoadsorción Enzimática , Citometría de Flujo , Expresión Génica , Técnicas de Transferencia de Gen , Inmunohistoquímica/métodos , Ratones , Ratones Endogámicos BALB C , Nucleósido Desaminasas/inmunología , Pruebas de Precipitina , ConejosRESUMEN
The Jijoye EBV strain is characterized by a substitution of 1.8 kb in the C-terminal part of the EBNA 2 gene compared to B95-8 or M-ABA virus. This made it possible to construct hybridization probes specific for M-ABA (type A) and Jijoye viruses (type B), which have been used to type the EBV genomes in 38 spontaneously established cell lines. Type A is more prevalent being found in 31 of 38 cases; type B virus was found in five cell lines (Jijoye, LY 67, QIMR-GOR, BL 16, and BL 29); and two cell lines, Daudi and EB-3, contained neither the M-ABA- nor the Jijoye-specific sequences. EBV type B appears to be less ubiquitous, since all type B isolates, including AG 876 virus, originated from Central Africa, La Réunion, and New Guinea. All the other cell lines, carrying EBV type A, were established from patients from Central Africa (4), North Africa (7), New Guinea (1), and Asia (6) and from white individuals (13). The restricted geographical localization of EBV type B in parts of the southern hemisphere and its similarity to herpesvirus papio (T. Dambaugh, K. Hennessy, L. Chamnankit, and E. Kieff (1984) Proc. Natl. Acad. Sci. USA 81, 7632-7636) could suggest that such viruses may have evolved by recombination of EBV with a related Old World monkey virus, alternatively, evolution of virus variants within the human species also being conceivable.