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PubMed1992

The prognostic value of cytometric DNA analysis in early stage tongue cancer.

In spite of a small size and seemingly localised properties T1 tongue cancer does recur after surgical treatment, locally and/or in regional lymph-nodes, in 30-40% of the patients, and 50% of patients with recurrent disease die because of their cancer. If these patients could be identified by analysis of relevant parameters on the primary biopsy reflecting the biological properties of the tumours more extensive treatment regimes could be given selectively. In 47 primary biopsy specimens from patients with T1N0M0 squamous cell carcinoma of the mobile tongue the aberration in cellular DNA content was significantly higher in the group of tumours which recurred after surgical treatment compared with the non-recurrent group. Tumours in females recurred more frequently than in males. No significant correlation between recurrence and grade of histological differentiation or tumour thickness could be found. Image cytometry DNA analysis provides an objective and reproducible assessment of the nuclear DNA content which could facilitate selection of adequate treatment strategies.

Adult↗

PubMed1990

Paternity testing: blood group systems and DNA analysis by variable number of tandem repeat markers.

Two recent paternity cases are reported. In the first case of paternity exclusion, deoxyribonucleic acid (DNA) restriction fragment length polymorphisms (RFLPs) on variable number of tandem repeat (VNTR) loci with multiple alleles were informative, as well as established systems of red blood antigens, red cell enzymes, serum proteins, and human leukocyte antigens. In the second case, in which both the alleged father and the first wife were deceased, the paternal genotype was determined by using genetic markers from the second wife and four children, which then were compared with the paternal alleles of the child in question, the plaintiff in this case. The high probability of paternity (0.999,998,7) made us conclude that the man probably was the actual father. The DNA analysis by VNTR probes appears to be quite valuable in the study of paternity cases.

Alleles↗

PubMed2002

Maternity testing using mitochondrial DNA analysis.

We examined the maternal relationship in two cases using mitochondrial DNA (mtDNA) analysis. The hypervariable region, HVI in the displacement loop (D-loop) region, was amplified and sequenced. Samples from both cases showed the nucleotide replacements of A to C at positions 16,182 and 16,183, and T to C at position 16,189, giving rise to a C-stretch from position 16,182 to 16,193, in which twelve Cs continued. We were unable to read the region after the C-stretch by forward sequencing in both cases, and it was thought that heteroplasmy was the reason for this. However, there were other identical nucleotide replacements in addition to the C-stretch in both cases. Additionally, nucleotide sequencing was used successfully to demonstrate complete maternal inheritance in all the offspring of eight families over two or three generations. We were therefore able to confirm the maternal relationship in the two present cases, and accordingly we consider that mtDNA sequencing is a very useful method for examining the maternal relationship, especially in cases where the father is not available.

Aged↗

PubMed1988

Molecular basis of beta thalassemia in south China. Strategy for DNA analysis.

The phenotype of beta thalassemia can be caused by over 40 different mutations. To set up a prenatal diagnosis program using DNA analysis, it is important to determine the type and frequency of mutation in a particular geographic area. We have delineated the molecular lesions that cause beta thalassemia in the Guangdong province of China, and found six mutations in four different haplotypes. The surprising finding that five of these mutations each occur in two different haplotypes suggests the occurrence of crossing over or gene conversion events at the beta-globin locus. The delineation of the haplotypes and mutations will permit the choice of the appropriate probes for prenatal detection of beta thalassemia in this part of China.

China↗

PubMed1999

[Taxonomic studies on Echinochasmus fujienensis and its related species by random amplified polymorphic DNA analysis and experimental infection].

AIM: To explore the identification and differentiation of Echinochasmus fujianensis, Echinochasmus japonicus(Jiangxi strain and Fujian strain), Echinochasmus liliputanus Anhui and Echinochasmus per foliatus Hubei. METHODS: Random amplified polymorphic DNA analysis (RAPD) and experimental animal infection were performed. RESULTS: 469 polymorphic DNA fragments were obtained by 28 primers from 4 Echinochasmus species and strains in Fujian, Anhui and Jiangxi. 20.8% and 97.6% of the fragments in Echinochasmus fujianensis were the same as those in Echinochasmus japonicus Fujian strain and in Echinochasmus liliputanus Anhui, respectively, 99.7% of the fragments were the same between Echinochasmus japonicus Jiangxi strain and Fujian strain. CONCLUSION: Echinochasmus fujianensis and Echinochasmus liliputanus Anhui are the same species. Echinochasmus fujienensis is an independent species different from Echinochasmus japonicus. Polyinfection of Echinochasmus fujianensis, Echinochasmus japonicus and Echinochasmus perfoliatus exist in all the 3 provinces, Hubei, Anbui and Fujian, of which Echinochasmus fujienensis is a dominant species.

Animals↗

PubMed1985

Prenatal diagnosis of hemoglobinopathies by DNA analysis.

Restriction site polymorphisms are normal inherited variations in DNA that can be readily detected by restriction endonuclease analysis. Currently, 17 such polymorphisms are recognized within a 60 kb (kilobase) stretch of DNA which includes the beta-globin gene complex. Because of their proximity to the beta-globin gene, often these restriction site polymorphisms can be used to predict inheritance of beta-globin variants that produce disease. For example, restriction site polymorphisms can be used for prenatal diagnosis for the large majority of couples at risk of having a child with beta-thalassemia. When each member of such a couple is heterozygous at one or more of these 17 sites, family studies are usually successful in determining which forms of the polymorphism are co-inherited with the beta-thalassemia genes in that particular family. Subsequently, study of fetal DNA isolated from amniocytes obtained by midtrimester amniocentesis or from chorionic villi obtained by first trimester chorion biopsy will reveal which DNA polymorphisms that fetus has inherited. By deductive reasoning one can then predict which beta-globin genes it has co-inherited. Because of the general nature of these polymorphisms, which are related to the beta-globin gene and its variants only because of their proximity on chromosome 11, they are potentially useful in the prenatal diagnosis of any beta-chain hemoglobinopathy. Some hemoglobinopathies (including alpha-thalassemia, sickle cell anemia, and some cases of beta-thalassemia) can be detected directly by DNA analysis. In these cases in utero diagnosis does not need to rely on restriction site polymorphisms, which require preliminary family studies and are not applicable in all at risk pregnancies. Recently, genetic probes, which are necessary for detecting restriction site polymorphisms, have been isolated for sequences of several genes whose protein products are important in blood coagulation. These include probes for all three genes whose polypeptide products combine to form the fibrinogen molecule as well as probes for the prothrombin, Factor IX, Factor VIII, and antithrombin III genes. Defects in these genes are expected to be the causes of afibrinogenemia, prothrombin deficiency, hemophilia B, hemophilia A, and antithrombin III deficiency, respectively. From experience with other genes, it is expected that restriction site polymorphisms within and/or flanking these genes will be found.(ABSTRACT TRUNCATED AT 400 WORDS)

Anemia, Sickle Cell↗

PubMed1990

The human aminopeptidase N gene: isolation, chromosome localization, and DNA polymorphism analysis.

DNA encoding the human aminopeptidase N (EC 3.4.11.2) gene (PEPN) was first isolated using rat cDNA probes and then used in Southern analysis of DNA from mouse-human somatic cell hybrids to assign this gene to the long-arm region (q11-qter) of human chromosome 15. This human genomic DNA probe detects a frequent DraIII polymorphism that is a useful marker for human chromosome 15.

Amino Acid Sequence↗

PubMed2003

Polymerase chain reaction-based methods of DNA methylation analysis.

DNA methylation is the main epigenetic modification in humans, and changes in methylation patterns play an important role in tumorigenesis. Hypermethylation of normally unmethylated CpG islands in the promoter regions often occurs in important tumor suppressor genes, DNA repair genes, and metastasis inhibitor genes. The changes of methylation status of various gene promoters seem to be a common feature of malignant cells and these changes can occur early in the progression process. Therefore detection of aberrant promoter hypermethylation of cancer-related genes may be useful for cancer diagnosis or detection of cancer recurrence. The purpose of this review is to provide a summary of the most commonly used techniques for the study of DNA methylation. Current scientific literature involving methylation detection methods was reviewed with an emphasis on polymerase chain reaction (PCR)-based detection methods. The current methodologies may be broadly classed into PCR-based methylation assays and non-PCR-based methylation assays. The problems and advantages of the different methods for detecting aberrant methylation are discussed. As the number of genes known to be hypermethylated in cancer is growing, the detection of aberrant promoter region methylation will be a promising approach for using DNA-based markers for the early detection of human cancers. Many techniques, especially PCR-based methylation assay techniques, make it practical to use these new methylation biomarkers in early cancer diagnosis.

Base Sequence↗

PubMed1996

Flow cytometric DNA analysis in endometriotic tissue compared to normal uterine endometrium.

Endometriotic tissue sometimes shows an invasive pattern. but the growth regulation of the tissue is insufficiently characterized. In a research programme on factors regulating endometriotic growth, the DNA ploidy status and S-phase fraction (SPF) were studied. Fresh-frozen endometriotic tissue from 14 women and endometrium from 11 of them were studied using flow cytometry. A clear diploid pattern was seen in most cases of endometriotic (8/14) and endometrial (8/11) samples. In the remaining cases the G0/G1 peak was broad and skewed, which might indicate a near-diploid cell population. To clarify this, a second group was studied, consisting of 29 formalin-fixed endometriotic samples from 22 women and endometrium from five of them. All these samples were diploid, with one having a broad G0/G1 peak. No convincing difference in SPF between endometrium and endometriotic tissue was found, as the calculations had to be handled with caution because of debris in many samples. Although the study of fresh-frozen samples gave some indications of differences in DNA ploidy status, flow of cytometric DNA analysis of formalin fixed samples of endometriosis showed a diploid DNA pattern in all samples. In conclusion, DNA flow cytometry did not show a convincing aneuploid DNA pattern in endometriotic tissue.

DNA↗

PubMed1988

A case of a lymphoproliferative disorder: usefulness of DNA analysis in diagnosis.

We describe a patient in whom a suppressor/cytotoxic T cell lymphoma was suspected on the basis of histology and immunohistochemistry. DNA analysis of specimens from three different lesions, using restriction enzymes Eco RI and Bam HI, showed the same rearrangement of the T cell receptor beta-chain genes. We consider this technique to be of value in the early diagnosis of malignant lymphoma when differential diagnosis from other lymphoproliferative disorders is difficult with conventional methods.

Aged↗

PubMed1993

Soft tissue vascular tumors. A flow cytometric DNA analysis.

BACKGROUND: The clinical behavior of soft tissue vascular tumors is difficult to predict on histologic grounds alone. METHODS: To assess the usefulness of DNA flow cytometry in predicting the biologic behavior of these tumors, the authors studied 51 soft tissue vascular tumors by DNA flow cytometry of paraffin-embedded tissue. RESULTS: All 20 capillary hemangiomas, one epithelioid hemangioma, two spindle cell hemangioendotheliomas, and two benign hemangiopericytomas had a diploid DNA content. Of the 20 patients with angiosarcomas, their ages ranged from 9-80 years (mean, 44.0 years), and the mean follow-up period was 16.5 months. Ten tumors (including two postmastectomy angiosarcomas) were cutaneous, six were deep soft tissues, and four were from other sites. These tumors were classified histologically into three types: sinusoidal (n = 10), capillary (n = 7), and mixed type (n = 3). Thirteen of the angiosarcomas (65%) were diploid, and seven (35%) were aneuploid. Three patients with angiosarcoma had lung metastases at initial diagnosis, and seven had them later in the course. Nine patients died of their disease within a period of 18 months after the initial diagnosis. All five patients with malignant hemangiopericytoma died of their disease. Three of these lesions had a diploid DNA content, and two had an aneuploid content. Although it was found that all benign and intermediate-grade malignant tumors were diploid, there were no significant correlations among histologic type, DNA ploidy, and clinical outcome in angiosarcomas and malignant hemangiopericytomas. CONCLUSIONS: Based on this study, DNA analysis has limited value in predicting the biologic behavior of malignant vascular tumors of the soft tissue.

Adolescent↗

PubMed1988

Alveolar soft part sarcoma. An immunohistochemical, cytologic and electron-microscopic study and a quantitative DNA analysis.

The type, differentiation and histogenesis of the tumor cells of alveolar soft part sarcoma (ASPS) have been analyzed in a series of ten cases by a light-microscopic, ultrastructural, immunohistochemical and cytologic investigation and quantitative DNA analysis. Four tumors deviated from ordinary ASPS: three were wholly or partly of the so-called pleomorphic variant of ASPS and a fourth tumor showed calcifications of the psammoma body type. The ultrastructural findings and immunohistochemical demonstration of desmin supported the hypothesis of a rhabdomyomatous differentiation and gave no support to epithelial (negative immunoreactions for cytokeratins, epithelial membrane antigen, HMFG-1 and -2, tissue polypeptide antigen (TPA] or neuroectodermal (negative for S-100 protein, glial fibrillary acidic protein, neurofilaments) differentiation. The negative immunoreactions for vimentin and myoglobin and the positive reaction for neuron specific enolase (NSE) do not exclude a rhabdomyomatous differentiation since in rhabdomyosarcomas the undifferentiated rhabdomyoblasts generally contain vimentin and the differentiated tumor cells contain myoglobin and rhabdomyosarcoma has previously been reported as being positive for NSE. The production of external lamina material peripherally in the tumor cell nests and around vessels in the vascular septa was demonstrated both ultrastructurally and by immunohistochemistry using antibodies against collagen IV and laminin. The cytologic appearance in smears obtained by fine-needle aspiration from a case of the pleomorphic variant showed some resemblance to that of a carcinoma. The seven tumors with an ordinary cell appearance were found to show a diploid DNA-distribution at a quantitative analysis performed on paraffin sections, while the three tumors wholly or partly of the pleomorphic type showed an additional tetraploid peak.

Adolescent↗

PubMed1997

DNA-ploidy abnormalities are a reflection of the metastatic potential of malignant melanoma. Microfluorometric DNA analysis.

Using DAPI (4',6-diamidino-2-phenylindole)-DNA microfluorometry, we examined the nuclear DNA-ploidy abnormalities of 15 primary malignant melanomas and their 20 corresponding metastases. They all presented the aneuploid DNA histographic pattern. When the DNA index value was calculated as the reflection of DNA-ploidy abnormalities, it was found to be significantly higher in the metastases (2.07 +/- 0.50) than in the primary tumors (1.76 +/- 0.50) (p < 0.01). Sixteen (80%) of the metastatic tumors had a higher DNA index value than their primary tumors, whereas the remaining four (20%) had a lower value. The difference in the DNA index values between the primary and metastatic tumors did not correlate to any other conventional prognostic variables (e.g. histologic type, level, and thickness). When we added 15 non-metastatic melanomas to the above 15 primary melanomas and evaluated the predictors for metastasis using multivariate stepwise logistic regression analysis, the DNA index value of the primary melanomas was found to be the most reliable risk factor. These results suggest that primary melanoma cell populations, having high DNA index values, are usually responsible for subsequent metastasis, and that hence, DNA-ploidy abnormalities of primary melanomas are likely to provide useful information for patient potential with regard to metastasis.

Adult↗

PubMed1997

Variability of DNA analysis by image cytometry. Bladder Tumor Marker Network.

Two laboratories equipped with CAS 200 (Becton Dickinson Image Cytometry Systems, San Jose, CA) instruments participated in this study of variability of DNA analysis of bladder tumor specimens. Formalin fixed paraffin embedded specimens were disaggregated and centrifuged onto microscope slides from ten bladder tumor specimens and two specimens of normal urothelium. Sources of variability considered were Specimen, Slide, Run, Laboratory, and Error. Slides were systematically scanned and 200 cells measured followed by the operator selecting 100 nuclei with abnormal morphology. DNA index (DI) and hyperdiploid fraction (HDF) were calculated from the DNA frequency distributions. For systematic sampling, 92% of the variability was due to Specimen indicating that differences in HDF values between specimens reflect biological differences. With selective sampling, only 67% of the variability in HDF is due to Specimen differences. Other factors, Laboratory, Error, and Laboratory x Specimen interaction each accounted for approximately 10% of the variability. Similarly variability of DI with selective sampling was also higher, and less specimen dependent than systematic sampling. It is important that sampling schemes and selection criteria be carefully documented in order to control variability. Enriched (or selective) sampling for abnormal cells has the potential to increase sensitivity but specimen classification based on these measurements must depend on determination of the frequency of such cells in the total population.

Analysis of Variance↗

PubMed1998

[Flow cytometric DNA analysis and MR imaging of salivary gland tumors].

We correlated the histopathology of 32 cases of salivary gland tumors with MR imaging and flow cytometric DNA analysis. All malignant tumors were invasive and/or had an ill-defined margin. Fifty-seven percent of the pleomorphic adenomas were shown as a high signal intensity area on T2 weighted images. Fifty-six percent of Warthin tumors were shown as a low or iso signal intensity area on T2 weighted images associated with the cystic portion. Sixty-seven percent of malignant tumors were characterized by DNA aneuploidy and/or a total percentage of the S-phase fraction plus the G2 + M fraction higher than 10%. It was less than 10% and the Ki-67 positive fraction was higher than 20% in all pleomorphic adenomas. The Ki-67 positive fraction was less than 20% in 78% of Warthin tumors. The histopathology of all malignant tumors, 86% of the pleomorphic adenomas, and 89% of Warthin tumors was correctly predicted by the combination of MR images and flow cytometry. We suggest that the MR images combined with flow cytometric analysis of fine needle aspiration-derived materials is useful in the pre-operative histopathologic diagnosis of salivary gland tumors.

Adenolymphoma↗

PubMed1984

Restriction endonuclease DNA analysis of Leptospira interrogans serovars icterohaemorrhagiae and hebdomadis.

Antigenic variants of Leptospira interrogans serovars copenhageni and hebdomadis were examined by bacterial restriction endonuclease DNA analysis with EcoRI, XhoI, SalI, BstEII, and HindIII as the digesting enzymes. The antigenic variants were stable cloned strains which had been cultivated in media containing homologous immune serum. One of the strains examined has been reported elsewhere (R. Yanagawa and J. Takashima, Infect. Immun. 10:1439-1442) as having an antigenic makeup which more closely resembles serovar kremastos than the serovar hebdomadis parent. The closely antigenically related but naturally occurring serovars icterhaemorrhagiae strain RGA and copenhageni strain M20 were examined in parallel. No differences could be shown between the hebdomadis parent and any of its mutants. Serovars copenhageni and icterohaemorrhagiae produced patterns which differed in the high-molecular-weight bands only. The Shibaura parent strain did not differ from copenhageni M20, but the Shibaura M1 strain differed from the other mutants and from icterohaemorrhagiae RGA in its high-molecular-weight bands.

DNA Restriction Enzymes↗

PubMed1994

Comparative DNA analysis by image cytometry and flow cytometry in non-small cell lung cancer.

To determine whether image cytometry (ICM) is advantageous for clinical DNA analyses of tumor cells, nuclear DNA contents measured by ICM were compared with those by flow cytometry (FCM), using 46 samples of non-small cell lung cancers. ICM was performed on smear specimens of fresh materials (f-ICM) and cell suspensions obtained from paraffin-embedded tumors (p-ICM). The same cell suspensions were also analyzed by FCM (p-FCM). Aneuploid rates/coefficient of variation (CV) of f-ICM, p-ICM, and p-FCM were 76.1/4.90, 71.7/5.01 and 60.9/5.31%, respectively. There was a high correlation in the DNA indices between p-ICM and p-FCM (r = 0.80). In the comparative DNA analysis, there were seven discordant samples. Six of them were estimated as aneuploid by p-ICM, but they were miscounted as diploid or undefinable (impossible) by p-FCM. This was caused by measuring condensed nuclei or debris. All "impossible" samples in p-FCM were squamous cell carcinoma with necrosis. In cell cycle analysis, the S and S+G2/M phase fractions in diploid samples were higher in p-ICM than those in p-FCM (P < 0.005), because the G0/G1 phase (2N) fraction presented by FCM was composed of cancer and non-malignant cells in diploid cancers. In ICM, they can be separately measured by means of morphological selection. These findings indicated that ICM is superior to FCM, especially for the practical DNA measurement of a few cancer cells and in the evaluation of the proliferation rates.

Adult↗

PubMed1998

Comparison of routine flow cytometric DNA analysis of fresh tissues in two laboratories: effects of differences in preparation methods and background models of cell cycle calculation.

Routine flow cytometric DNA analysis was compared in two laboratories by using matched fresh-frozen breast cancer and soft tissue sarcoma biopsy specimens. Laboratory I applied the Vindelöv preparation method and an exponential background subtraction algorithm in the cell cycle calculation. Laboratory II used the Formalin-protease preparation technique and the sliced-nuclei background model. The results of the ploidy analysis showed good agreement between the two laboratories; however, the results of the cell cycle analysis showed considerable systematic differences between labs. Laboratory I obtained significantly lower values of S-phase fraction and higher values of G2-phase fraction than laboratory II. To explain these discrepancies, the effects of differences in the preparation methods and background subtraction algorithms were studied. The Vindelöv preparation method yielded higher debris and aggregation levels than the Formalin-protease technique and tended to give higher %S and %G2 values. When the two background models were used in the same histograms, the exponential background model tended to give %S values distinctly lower than and %G2 values almost identical to those obtained with the sliced-nuclei algorithm. The sum of these effects accounts for the observed inter-laboratory discrepancies. Different from the sliced-nuclei fit, the exponential background fit often did not accommodate to the original data in the <2c histogram region and resulted in a considerable inter-operator variability of %S calculation in histograms with <5% S. When aggregate correction was added to the sliced-nuclei algorithm, the differences between %S values in histograms from the two laboratories almost disappeared.

Algorithms↗