A rare cause of mediastinal expansion with a massive pleural effusion.

The authors present a case of a 53 year old woman, who was admitted to hospital because of an unusual cause of massive pleural effusion. During diagnostic examination the mediastinal propagation of the pancreatic pseudocyst was discovered as a complication of the chronic calcifying pancreatitis. The patient was operated on and the pseudocyst was resolved by Roux-en-Y cystjejunostomy. The diagnostics and treatment of this unusual pancreatic pseudocyst spreading is discussed.

[When is it useful to look for TP53 germline gene mutations in families of oncology patients?]. / Kdy má smysl hledat v rodinách onkologických pacientu zárodecné mutace v genu TP53?

BACKGROUND: The Li-Fraumeni syndrome is a relatively rare familial cancer syndrome associated with germline mutations in the tumour-suppressor gene TP53. Members of affected families can suffer from a wide variety of tumours. Identification of a germline TP53 mutation is particularly important in families of patients affected by one of several characteristic types of tumours. METHODS AND RESULTS: The data used for the distribution analysis of mean age at the cancer diagnosis in TP53 mutation carriers and in the Czech population were extracted from a database of 176 families (469 cancers in 346 patients) with germline TP53 mutations and from Czech statistical data of cancer incidence in years 1994 to 1998 (UZIS). The comparison of the age distribution of the relative tumour incidence in these two groups clearly separated childhood adrenocortical sarcoma, rhabdomyosarcoma, osteosarcoma and brain tumour patients. In their families genetic counselling should be recommended. CONCLUSIONS: Patients with low age at diagnosis of these tumours, particularly patients with additional personal or family history of malignancy, should be considered as potential TP53 mutation carriers. It should be relevant not only for the treatment and follow-up of the patients but also for preventive measures aimed at other members of their families.

The expression of PAX5, p53 immunohistochemistry and p53 mutation analysis in superficial bladder carcinoma tissue. Correlation with pathological findings and clinical outcome.

OBJECTIVES: The expression pattern of PAX5 in the tissue of superficial bladder transitional cell carcinoma (TCC), its prognostic value and its correlation with p53 immunohistochemistry and p53 mutation analysis were evaluated. METHODS: Study comprised 61 patients with histologically confirmed superficial bladder TCC. Expression level of PAX5 mRNA was investigated using reverse transcriptase-polymerase chain reaction (RT-PCR) and determined semiquantitatively. The presence of p53 mutations was determined by SSCP and confirmed by direct sequencing. The p53 immunohistochemistry was performed with DO1 antibody and semiquantitatively evaluated using HSCORE (HS) method. As the control group for the evaluation of the PAX5 expression served 8 men with benign prostatic hyperplasia. RESULTS: PAX5 expression was found in 50 patients with bladder TCC but in no patient from the control group. Its quantity however correlated neither with the stage nor with the grade of the tumor. P53 mutation was confirmed only in 1 patient with pTaG2 tumor in exon 5 (deletion of proline 128). On the contrary, positive immunohistochemical staining of p53 was detected in most patients. Using the cutoff value of HS 200, 56.9% of patients showed p53 overexpression. Quantity of p53 immunochistochemical positivity did not correlate with the quantity of PAX5 expression. Using the cutoff values of HS 200 for p53 and of 0.2 for PAX5, 7 of 8 patients with future progression had p53 and 4 had PAX5 overexpression respectively. CONCLUSION: The expression of gene PAX5 is a frequent event in superficial TCC of the bladder.

Methylation changes in promoter and enhancer regions of the WT1 gene in Wilms' tumours.

Although the WT1 gene has been implicated in the aetiology of Wilms' tumour, mutations in WT1 are found only in minority of the tumours. DNA methylation of regulatory elements represents another possibility of modulation of gene expression. We studied methylation in the promoter and enhancer regions of the WT1 gene in 34 Wilms' tumour patients by the polymerase chain reaction on HpaII-digested DNA and by the bisulphite method. No methylation was detected in the promoter region in either tumour or normal kidney or blood DNA samples. In contrast, a HpaII site in the enhancer region was at least partially methylated in normal kidney and blood DNA samples and in about one-third of the tumours, while the majority of tumours showed no methylation. The differential methylation in the enhancer region of the WT1 gene may indicate that methylation of this element can play a role in the regulation of this gene.

[DNA methylation and neoplasms]. / Metylace DNA a nádorová onemocnení.

DNA methylation and acetylation of histone proteins represent two global mechanisms controlling the gene expression. DNA methylation profiles alter during the development of the organism and during progression of neoplasia. Three types of alterations of the DNA methylation profiles were observed in the tumor cells: hypomethylation, hypermethylation and the loss of imprinting. Beside the intra-gene mutation and the heterozygosity absence, DNA methylation can be understood as the third mechanism of tumor-suppressor gene inactivation in the genesis of neoplasia. Our review article brings recent findings and hypotheses on the role of DNA methylation in the carcinogenesis and its possible application in the diagnostics and therapy of the malignant proliferation.

Sternobronchial fistula--uncommon complication after coronary surgery (a case report).

The authors describe a case of a 46-year-old man with ischemic heart disease who underwent coronary surgery. After some time span an inflamed wound, several skin fistulae and the system of substernal fistulae appeared. One of these fistulae communicated with the left bronchial tree.

Oncogene amplification and expression in pediatric solid tumors.

Oncogene amplification and expression and their mutual relationship was analyzed in 92 pediatric tumors by Southern and Northern blot hybridization with N-MYC, ERB A, ERB B, N-RAS and Shb probes. Amplification and overexpression was associated with more advanced clinical stages of tumor, especially in neuroblastomas, rhabdomyosarcomas and ganglioneuroblastomas. The most frequent alteration observed was N-MYC amplification together with overexpression. N-RAS amplification was not detected, while the overexpression of this oncogene was found in 3 cases. Neither amplification nor overexpression was revealed in any specimen of hepatoblastoma or hepatocellular carcinoma. We suggest that oncogenes overexpression provides more accurate prognostic information than amplification.

Two Li-Fraumeni syndrome families with novel germline p53 mutations: loss of the wild-type p53 allele in only 50% of tumours.

We describe two Li-Fraumeni syndrome families. Family A was remarkable for two early childhood cases of adrenocortical tumours, family B for a high incidence of many characteristic cancers, including a childhood case of choroid plexus tumour. Using direct sequencing, we analysed exons 5-9 of the p53 gene in constitutional DNA of individuals from both families and found two novel germline mutations in exon 5. In family A, we detected a point substitution in codon 138 (GCC to CCC), which resulted in the replacement of the alanine by a proline residue. Family B harboured a single-base pair deletion in codon 178 (CAC to -AC), resulting in a frameshift and premature chain termination. Three out of six tumours examined from both families, a renal cell carcinoma, a rhabdomyosarcoma and a breast cancer, showed loss of heterozygosity and contained only the mutant p53 allele. The remaining three neoplasms, both adrenocortical tumours and the choroid plexus tumour retained heterozygosity. Immunohistochemistry with anti-p53 antibody confirmed accumulation of p53 protein in tumours with loss of heterozygosity, while the remaining tumours were p53 negative. These results support the view that complete loss of activity of the wild-type p53 need not be the initial event in the formation of all tumours in Li-Fraumeni individuals.

A database of germline p53 mutations in cancer-prone families.

We created a comprehensive database covering all published cases of germline p53 mutations. The current version lists 580 tumours in 448 individuals belonging to 122 independent pedigrees. The database describes each p53 mutation (type of the mutation, exon and codon affected by the mutation, nucleotide and amino acid change), each family (family history of cancer, diagnosis of Li-Fraumeni syndrome), each affected individual (sex, generation, p53 status, from which parent the mutation was inherited) and each tumour (type, age of onset, p53 status-loss of heterozygosity, immunostaining). Each entry contains the original reference(s). The database is freely available and can be obtained from http://www.lf2.cuni.cz

[Tumor suppressor genes]. / Tumor supresorové geny.

The main role of tumour suppressor genes is the inhibition of cell proliferation. Somatic mutations in these genes are found frequently in sporadic tumors. Germ line mutations in tumour suppressor genes are responsible for hereditary cancer syndromes. In a carrier of such a germ line mutation, a somatic mutation or loss of the remaining functional copy of the gene is sufficient for the complete loss of function of the tumour suppressor. Therefore the carriers of germ line mutations have a high risk of developing malignancies. Many tumour suppressor genes have been cloned and characterized recently and many others are intensively searched for. Protein products of these genes serve different cellular functions and many of them directly participate in the cell cycle control. The characterization of tumour suppressor genes is important both for the understanding of processes of carcinogenesis and for practical use in the diagnostics, prognostics and therapy of tumours.

[A method for detection of germinal mutations in the p53 tumor suppressor gene]. / Metodika detekce zárodecných mutací v tumor supresorovém genu p53.

BACKGROUND: The tumour suppressor gene p53 is exhibits somatic mutations in a high proportion of human tumours. In addition, there are cancer families suffering from the Li-Fraumeni syndrome, the members of which carry germ line mutations in this gene. The carriers of the p53 germ line mutations have a high risk of developing tumours. The genetic diagnosis of carriership of the mutation in the tumour family members is important for preventive measures and for eventual tumour therapy modification. METHODS AND RESULTS: We have developed a method for the detection of germ line mutations in the p53 gene based on non-radioactive SSCP and direct sequencing of PCR products. We have proved the efficiency of the method by finding known mutations in eight tumour cell lines. In our collection of tumour families we have detected polymorphisms in exons 4 and 6 of the p53 gene. In one family which conformed to the criteria of the Li-Fraumem syndrome we have found a novel germ line mutation in exon 5. CONCLUSIONS: The method developed by us is very simple and sensitive. The germ line mutations in the p53 gene are very rare.

[Amplification of oncogenes in solid tumors in children]. / Amplifikace onkogenu v detských solidních nádorech.

BACKGROUND: The objective of the work was detection of amplification of oncogenes N-MYC, N-RAS, C-ERB A, C-ERB B and adaptor tyrosine kinase Shb in a group of 92 child age tumours in an attempt to reveal clinical and histopathological associations. METHODS AND RESULTS: Amplifications of oncogenes were detected by means of Southern's transfer, hybridization with labelled probes and densitometric evaluation. Amplification of the N-MYC oncogene in child tumours can be considered a manifestation of progression of the disease with an adverse prognosis, in particular in neuroblastomas, where it corresponds also with the adverse histological finding. In a group of sarcomas N-MYC amplification was detected in advanced clinical stages, while in malignant lymphogranulomas of the Hodgkin type it was not found. In Wilms tumour it was detected sporadically. Amplifications of oncogenes ERB A and ERB B are rare, amplifications of the oncogene RAS were not observed. Coamplifications characterized progression of the disease, in case of neuroblastoma even very short survival. In hepatic malignancies oncogene amplification was not found even in advanced stages. CONCLUSIONS: Oncogene amplification characterizes progression in a number of child tumours and its application in clinical oncology is prognostically useful.

[Oncogenes and the malignancy process]. / Onkogeny a malignizacní proces.

An important group of genes for the development of neoplastic diseases are, in addition to tumour suppressor genes, protooncogenes. The latter are highly preserved genes present in a similar sequence in the cell genomes of different species (yeasts - man). They encode components of biochemical signalling pathways by which external mitotic signals stimulate cell proliferation and products which inhibit cell differentiation. The result of activation of protooncogenes into oncogenes (mutations, chromosomal rearrangements, amplifications, viral insertions, insertion mutagenesis) is in particular hyperstimulation of cells resulting in uncontrolled proliferation. Mutations are of the dominant type, elimination of one allele leads to the transformation of a protooncogene into an oncogene. Oncogenes are classified with regard to the transmission level of the mitogenic signal on which they act. Originally they were detected in the genome of oncogenic viruses. However, they do not form their constant and specific constituent, the virus acts as a vector which transmits cellular protooncogenes (or oncogenes) during the reproductive cycle from one cell to another. The activity of various types of oncogenes is the necessary prerequisite for the genesis and development of various neoplastic diseases. Detection of oncogene alterations provides in some instances important diagnostic, prognostic and therapeutic findings.

Construction of a transcription map of a 300 kb region around the human G6PD locus by direct cDNA selection.

A transcription map covering a 300 kb region around the G6PD gene in the human Xq28 region was constructed by the direct cDNA selection method and the analysis of the resulting region-specific enriched cDNA sublibrary. Seven new genes and two loci of endogenous retrovirus HERV-K were identified. The distribution of the genes across the region is strongly non-uniform and follows the non-uniform distribution of GpG islands in the area. While one of the novel genes was found to be highly homologous to bovine smg p25A GDP-dissociation inhibitor, the remaining genes did not detect any homology to known genes. The analysis of region-specific cDNA sublibraries represents a simple, rapid and efficient tool for the generation of a regional transcription map.

Direct selection of DNA sequences conserved between species.

An essential requirement in the analysis of genomes is the identification of functionally important sequence elements, which are often evolutionarily conserved. We describe here the development of a novel procedure for the selective isolation of conserved sequences which is based on hybridization of PCR-amplifiable DNA fragments from the whole genome of one species to biotinylated DNA from a genomic region of another species. The interspecies DNA hybrids are immobilized and the PCR-amplifiable DNA fragments are eluted, amplified and after further hybridization-amplification rounds cloned. This method was used for the generation of sublibraries of conserved sequences from mouse and pig DNA from regions corresponding to cosmids from the human Xq28 region. Mouse and pig homologs of sequences containing exons of known human genes, as well as exons from novel genes have been identified.

[Use of DNA analysis in medicine]. / Pouzití analýzy DNA v lékarství.

Advances in genetic engineering influence to an increasing extent a number of medical disciplines. DNA analysis can be used in the diagnosis of hereditary diseases, in investigations of malignant processes, in forensic medicine and for detection of infectious pathogens. Two main methodical approaches to DNA analysis, Southern's method and procedures based on primer directed enzymatic amplification of DNA by the PCR method, resolve the complicated detection of slight changes in the vast volume of human genetic information. Cystic fibrosis may serve as an example of a serious hereditary disease the diagnosis of which improved greatly after introduction of DNA analysis. The diagnosis of this disease is nowadays possible by direct analysis of mutations and indirectly by investigations of the link between the disease and DNA polymorphisms.

[Strategies in prenatal diagnosis of cystic fibrosis after the introduction of DNA analysis. Initial experience]. / Strategie prenatální diagnostiky cystické fibrózy po zavedení analýzy DNA. Prvé zkusenosti.

The authors describe their experience with the prenatal genetic diagnosis of cystic fibrosis (CF), using DNA analysis in the first trimester of pregnancy in three families with a 25% risk of CF. The authors examined polymorphisms of probes J3.11, met D, met H, KM-19 and XV-2c. All families were fully informative when one or two probes were used. In two families the development of unaffected children--carriers of the gene for CF was proved. In one of these foetuses in the 17th and 21st week false pathological values of microvilillous enzymes were assessed. With regard to this possibility the authors do not recommend to supplement the DNA analysis in the first trimester by biochemical examination of amniotic fluid. The results were confirmed by delivery of unaffected children. In one family DNA analysis revealed the development of an unaffected homozygote, the pregnancy was, however, terminated by a miscarriage. In women with an increased risk of abortion the authors recommend therefore to make the molecular genetic examination during the second trimester from amniotic fluid cells.

[Experience with rapid molecular genetic diagnosis using the polymerase chain reaction]. / Zkusenosti s metodou rychlé molekulárne genetické diagnostiky polymerázovou retezovou reakcí.

The authors give an account of their experience with 1000 amplifications of DNA by the polymerase chain reaction for the prenatal diagnosis of cystic fibrosis. The method is demonstrated on examples of examinations of the informativity value and prenatal diagnosis in the first trimester of pregnancy in families with a 25% risk of cystic fibrosis, using J 3.11 (Msp I), met H (Msp I), KM 19 (Pst I), CS 7 (Hha I), Mp6d9 (Msp I), XV 2c (Taq I) probes. The authors summarize methodical check-up and safety measures to ensure the reliability of diagnoses made by the PCR method.

The experience with the foetal diagnosis of the cystic fibrosis in the second and first trimester.

The amniotic fluid activity of gamma glutamyl transpeptidase (GGT), leucine aminopeptidase (LAP) and alcaline phosphatase (AP) and disacharidases was examined in 66 pregnancies with the risk of cystic fibrosis (CF) in the 17th-21st weeks of gestation. So far 28 pregnancies continue. The prenatal diagnosis was confirmed in all so far delivered children or aborted foetuses if the GGT activity was higher than 400 U/1 (10th percentile) or lower than 190 U/1 (3rd percentile) in the 17th-18th weeks. The results of other microvillar and ultrasound examinations were consistent with it. From 3 pregnancies with GGT activity in the range of 3-5 percentiles and abnormal activities of other microvillar enzymes, the CF was confirmed only in one aborted foetus with meconium ileus and with abnormal ultrasound examination. In other 2 pregnancies with normal ultrasound, healthy children were delivered. In 3 pregnancies with the GGT in the range of 5-10 percentiles and abnormal other microvillar enzymes, one false negative GGT and ultrasound examination was disclosed. The other 2 aborted foetuses did not exhibit the signs of CF in necropsy examinations. The meconium ileus was found in 2/4 of aborted foetuses with GGT lower than 3 percentiles, abnormal activities of other microvillar enzymes and abnormal ultrasound examination. The ultrasound examination was correct in 2/10 of pregnancies with GGT lower than 3 percentiles or abnormal activities of other microvillar enzymes. The GGT examination in 19th-21st weeks provided similarly reliable diagnostic results. The importance of fetal karyotyping and ultrasound elimination of other severe congenital anomalies is pointed out for critical interpretation of microvillar enzyme activities testing.(ABSTRACT TRUNCATED AT 250 WORDS)

The rapid molecular genetic diagnosis of cystic fibrosis by polymerase chain reaction: an experience report.

The authors report their experience with about two thousand DNA amplifications by polymerase chain reaction (PCR) in prenatal diagnosis of cystic fibrosis. The method is demonstrated on examples of diagnostic informativity and prenatal diagnosis examination in a family at 1 in 4 risk of the disease using closely CF-linked diagnostic polymorphisms: J3.11/MspI, MetH/MspI, CS7/HhaI, KM19/PstI, Mp6-d9/MspI and XV2c/TaqI, PCR methodology and safety precautions are discussed.