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PubMed1989

[Flow cytometric DNA analysis in rectal carcinomas].

The cellular DNA content was measured with flow cytometry from paraffin-embedded material in 329 patients and metastatic tumors of the liver from the rectum in 11 patients. The classification of the DNA ploidy pattern is as follows: A stem cell peak with a DNA index of 0.9-1.1 is defined as DNA diploid tumor and DNA aneuploid tumor is that with a DNA index greater than or equal to 1.1. There was a good correlation of DNA indices (r = 0.997) obtained from flesh and corresponding paraffin-embedded specimens. It is concluded that accurate determination of DNA index from paraffin-embedded materials is possible in the majority of cases. DNA ploidy of primary tumor cells correlated with clinicopathological findings such as lymphatic invasion, vascular invasion, lymph node metastasis and hepatic metastasis (p less than 0.01), but did not correlate with extramural carcinoma invasion. The cumulative survival rate (Kaplan-Meier) of curatively resected rectal carcinomas was worse in DNA aneuploid than in DNA diploid tumors (p less than 0.01). These observation showed that the determination of DNA ploidy in rectal carcinomas may prove to be of prognostic value.

Aneuploidy↗
PubMed1993

[Clinical significance of flow cytometric DNA analysis in patients with rectal cancer].

DNA content was measured by flow cytometry using paraffin-embedded material from 148 primary rectal cancers and 10 distant metastatic lesions. DNA ploidy pattern was classified into the following three groups in terms of the DNA index: 1.00 was defined as the diploid group (DP), 1.00 < DNA index < 1.60 as the low-aneuploid group (LAP) and > or = 1.60 as the high-aneuploid group (HAP). Primary carcinomas were DP in 26.4%, LAP in 36.5% and HAP in 37.1%. The DNA ploidy pattern of the primary tumor correlated well with clinicopathological findings such as depth of invasion, lymphatic invasion, pathological stage, metastasis to distant organs and curability of the tumor. In patients with HAP tumor after curative operation, the recurrence rate (21.6%) in distant organs was significantly higher than those with DP tumor (2.8%) and LAP tumor (6.4%) [p < 0.05]. Two LAP patients and 8 HAP patients with distant metastatic disease had the same DNA ploidy pattern HAP in the metastatic lesions. These data indicate that tumor DNA ploidy patterns classified into three groups in rectal cancer may play an important role in predicting prognosis, including distant metastasis.

DNA, Neoplasm↗
PubMed2000

Intraspecific variation in two species of Rhizomucor assessed by random amplified polymorphic DNA analysis.

Twenty-three Rhizomucor isolates were characterized by random amplified polymorphic DNA-PCR (RAPD-PCR) with 10-bp oligonucleotide primers. These data were used for numerical analyses to obtain information on the intraspecific genetic polymorphism of Rhizomucor species. The genetic variability in Rhizomucor pusillus and Rhizomucor miehei isolates was found to differ; the latter revealed less intraspecific polymorphism. The different levels of genotypic diversity suggest a correlation with the different forms of mating behaviour of these species. Rhizomucor tauricus displayed amplification patterns similar to those of the investigated R. pusillus strains, reinforcing the assumption that R. tauricus does not represent a separate species. Characteristic RAPD markers allowing PCR-based species identification of Rhizomucor isolates were determined.

Animals↗
PubMed2003

Ancient DNA analysis of the delta F508 mutation.

When working with highly degraded DNA, validating the results of a slightly polymorphic system always complicates the analysis because of the difficulties in recognizing contamination and artifacts. Recognition can be greatly simplified by employing a multiplex reaction that coamplifies the fragments together with several highly polymorphic markers, for instance, short tandem repeats. In this work, we successfully included newly designed oligonucleotide primers for the detection of delta F508, the most frequent mutation causing cystic fibrosis, in the commercial AmpFlSTR Profiler Plus PCR Amplification Kit (PE Applied Biosystems). This coamplification enabled us to test the hypothesis of a heterozygote advantage associated with cystic fibrosis-specifically, higher resistance to toxin-mediated diarrheas--in a Sicilian skeletal sample of individuals who died in a cholera epidemic in 1837. The proposed method should also be suitable for the genetic characterization of other slightly polymorphic loci tested on human and animal ancient DNA; it should permit simple authentication of results by comparing the fingerprints obtained from independent amplifications repeated several times.

Cystic Fibrosis↗
PubMed1986

Quantitative nuclear DNA analysis of human ovarian adenocarcinoma: compared before and after chemotherapy and correlated with clinical response.

Quantitative DNA measurements on 18 human ovarian adenocarcinomas were made by computerized image analysis. The DNA content of the tumor cells was measured on specimens of tumor obtained at the initial diagnostic surgery and at second-look surgery after treatment with chemotherapy. The mean DNA content of the specimens and the ploidy pattern of the tumor cells were determined. With the exception that borderline tumors had near normal ploidy patterns and mean DNA content, there was no consistent correlation between the stage of disease, grade, or histiologic character of the tumor and either the DNA content or ploidy pattern. But it was noteworthy that all three of the patients who had complete responses (negative second-looks), also had tumors with DNA content and ploidy patterns near triploid. When the ratio of mean DNA content before and after chemotherapy was determined for each ploidy group, there was an apparent correlation between this ratio and clinical status of the patient 10 month after chemotherapy. That is, patients with low ploidy tumors and high DNA content ratio (greater than 1.25) had a better prognosis than patients with high ploidy tumors and lower DNA content ratios (less than 1.25). Thus, although the mean DNA content of the tumor at the initial surgery was not in itself of sufficient prognostic value, when the mean DNA content of the tumor after chemotherapy is also known, an accurate picture of the patients clinical response could be determined.

Adenocarcinoma↗
PubMed1992

Flow cytometric DNA analysis and clinical correlations in multiple myeloma.

DNA content of both bone marrow and peripheral blood mononuclear cells was measured by flow cytometric analysis in 46 patients with untreated multiple myeloma and 15 patients with benign monoclonal gammopathy to clarify further the incidence and clinical correlations of DNA aneuploidy. Aneuploidy was detected in the bone marrow of 25 multiple myeloma patients (54%) but in only one benign monoclonal gammopathy patient (7%), who developed multiple myeloma 34 months later. Thus DNA aneuploidy is considered rare in benign monoclonal gammopathy. In two multiple myeloma patients, DNA aneuploidy was detected also in blood, indicating circulating myeloma cells. The light chain of the M component was more frequently lambda in the diploid and kappa in the aneuploid group. Most of the patients with only light chain secretion were DNA aneuploid. Multiple myeloma patients with DNA hypodiploidy (7%), biclonal aneuploidy (4%), or DNA aneuploidy detectable in blood (4%) did not respond to therapy with melphalan and prednisone. Survival was not influenced by DNA content. No DNA aneuploidy was detected in the bone marrow or the peripheral blood of 26 patients with chronic lymphocytic leukemia or two patients with Waldenström's macroglobulinemia.

Adult↗
PubMed2001

Phyllodes tumors of the breast: correlation of nucleolar organizer regions with histopathological malignancy grading, flow cytometric DNA analysis and clinical outcome.

To examine whether nucleolar organizer regions detected by argyrophilia (Ag-NOR counts) can be used as a prognostic indicator in phyllodes tumors of the breast, and to compare its usefulness with that of DNA flow cytometric analysis, 28 cases of breast phyllodes tumors (including 15 benign, two borderline and 11 malignant tumors) were subjected to Ag-NOR staining and counting as well as DNA flow cytometric analysis. S-phase fraction and DNA ploidy analysis showed useful trends for improving outcome predictions in malignant phyllodes tumors. However, high Ag-NOR counts were significant in predicting survival status (P = 0.013) and reached near statistical significance in predicting survival times (P = 0.07). In predicting survival status, results for Ag-NOR counts were significantly better than those for ploidy analysis (P = 0.02) and S-phase fraction (P < 0.01). Only S-phase fraction was significantly predictive of survival times (P = 0.025). It is concluded that Ag-NOR counts and DNA flow cytometric analysis, easily performed using paraffin sections, give information that can improve predictions made by histopathological classification. Ag-NOR counts are significant in predicting survival in the presence of histopathological features of malignancy.

Adolescent↗
PubMed1992

Nuclear-encoded tobacco chloroplast ribosomal protein L24. Protein identification, sequence analysis of cDNAs encoding its cytoplasmic precursor, and mRNA and genomic DNA analysis.

Using a Nicotiana tabacum leaf cDNA library in the expression vector lambda gt11, two cDNAs encoding the full-length precursor polypeptide (M(r) 20,696) of tobacco chloroplast ribosomal protein L24 were identified and sequenced. These cDNAs encode a mature protein of 146 amino acids (M(r) 16,418) with a transit peptide of 41 amino acids (M(r) 4,278). The mature tobacco L24 protein has 78, 65, 45, and 35% sequence identity with ribosomal proteins L24 of pea, spinach, Bacillus subtilis, and Escherichia coli, respectively. The transit peptide of tobacco L24 is 54 and 57% identical with that of L24 chloroplast ribosomal proteins of pea and spinach, respectively. An expressed beta-galactosidase:L24 fusion protein, bound to nitrocellulose filters, was used as affinity matrix to purify monospecific antibody to L24 protein. Using this monospecific antibody protein L24 was identified among high performance liquid chromatography (HPLC)-purified tobacco chloroplast ribosome 50 S subunit proteins. The predicted amino terminus of the mature L24 protein was confirmed by partial sequencing of the HPLC-purified L24 protein. Northern blot analysis revealed a single mRNA band (0.85-0.90 kilobase) corresponding in size to full-length L24 cDNA. The presence of multiple genes for L24 is suggested by Southern blot hybridization and characterization of two cDNAs for L24 which only differ in their 3'-noncoding sequences.

Amino Acid Sequence↗
PubMed1988

Techniques for mitochondrial and ribosomal DNA analysis of anopheline mosquitoes.

Methods are described for the isolation of mitochondrial and total cellular DNA from mosquitoes. The mitochondrial and ribosomal DNA restriction patterns could be detected in the total DNA of an individual mosquito by the use of cloned probes. DNA restriction analysis may prove to be a useful alternative to isozyme electrophoresis for the study of insect population genetics.

Animals↗
PubMed1992

The contribution of DNA analysis to carrier detection and prenatal diagnosis of hemophilia A and B.

Developments in DNA technology have provided a novel means of carrier detection and prenatal diagnosis of hemophilia A and B. The collection of a large set of data has enabled us to evaluate the present feasibility and reliability of a diagnosis at the gene level and its contribution to methods already available. Since 1984, 533 potential and obligate carriers belonging to 170 families with hemophilia have been referred to us. By the combined use of pedigree analysis, coagulation assays, and DNA (RFLP) analysis, certainty about the carrier status has been markedly increased for the potential carriers. Although RFLP analysis revealed the possible origin of the mutation in many families with an isolated patient, uncertainty remained for quite a number of their female relatives because of the possible occurrence of germline mosaicism. Forty-one women requested prenatal diagnosis during one or more pregnancies. The short time interval between pregnancies, even after abortion of an affected fetus, proved that first-trimester prenatal diagnosis has become an acceptable option for women at risk. Recently, efficient methods for direct identification of mutations have been developed, and they may allow a definite diagnosis for all families with hemophilia in the near future.

DNA↗
PubMed1996

Germline mutations of the RET proto-oncogene in pedigree with MEN type 2A: DNA analysis and its implications for pediatric surgery.

To assess the feasibility of screening for multiple endocrine neoplasia type 2A (MEN 2A), the authors used DNA sequence analysis to evaluate the RET proto-oncogene in a kindred with MEN 2A. The kindred consisted of 95 members (1 to 79 years of age) and their spouses, and spanned five generations. Genomic DNA was extracted from peripheral blood lymphocytes or lymphoblastoid cell lines established from the family members, and the RET gene was amplified by polymerase chain reaction (PCR) using RET-specific primers (10q 11.2) and was sequenced. Periodic endocrine screening also was performed, by measuring the plasma calcitonin concentration after provocation with pentagastrin (0.5 microgram/kg intravenously) to assess its reliability for detecting the associated neoplasms. Nineteen patients were confirmed to have MEN 2A by medical records or the screening program. The DNA sequence of the PCR products from clinically established MEN 2A patients showed a mutation at codon 634 (TGC-->CGC) that resulted in an amino acid change from cysteine to arginine. Endocrine screening tests showed that six other family members had a mutated RET protooncogene. DNA sequencing can detect high-risk cases at a preclinical stage of the disease. The establishment of mutated MEN 2A gene carriers allows pediatric surgeons to consider total thyroidectomy at a very early stage of neoplasm development (C-cell hyperplasia) or even prophylactically.

Adolescent↗
PubMed1991

Improved sensitivity and resolution in the flow cytometric DNA analysis of human solid tumor specimens. Use of in vitro fine-needle aspiration and uniform staining reagents.

Twenty-five unselected fresh colon or breast tumors were studied to identify specific components of sample preparation, sample staining, and flow cytometer operation affecting the sensitivity of DNA stemline analysis. Solid tumors were disaggregated using both a published method for mechanical/enzymatic whole cell (M/EWC) isolation and a fine-needle aspiration (FNA) technique. Staining FNA samples with CycleTEST propidium iodide reagents demonstrated improved sensitivity in the recognition of near diploid and near tetraploid aneuploid populations: 9 of 20 resolvable aneuploid DNA stemlines identified in FNA suspensions were not detected or clearly resolved in M/EWC preparations. These results suggest that previously reported discordances between flow and static image cytometry in the recognition of near tetraploid DNA stemlines may be related to inherent limitations of M/EWC. The in vitro FNA technique, when compared to M/EWC, may yield increased sensitivity and precision in clinical DNA stemline analysis when using fresh, unfixed, solid tumor specimens.

Aneuploidy↗
PubMed1988

Predictive value of flow cytometric DNA-analysis on fresh retinoblastoma tissue.

The cellular DNA content and the distribution of tumour cells in different phases of the cell cycles has been analysed in 8 consecutive enucleated eyes with retinoblastoma. All tumours had abnormal ploidy levels. The analysis did not reveal any specific pattern in 2 tumours which had metastasized compared to 6 local tumours. The flow cytometric analysis alone or in combination with histopathology appeared not to improve the classification of large retinoblastomas.

Child, Preschool↗
PubMed1987

Comparison of cytologic composition with microfluorometric DNA analysis of the glioblastoma multiforme and anaplastic astrocytoma.

The DNA content of selected areas of 16 glioblastoma multiforme predominantly composed of small anaplastic cells was investigated. These findings were contrasted with those of five cases of anaplastic astrocytomas. The microfluorometric determination of DNA was performed on mechanically isolated single cells stained by the Feulgen method, obtained from selected areas of the neoplasms in paraffin blocks. Twelve of 16 cases of glioblastoma multiforme had a main population that was diploid-near diploid. Although four of the five anaplastic astrocytomas disclosed a diploid-near diploid main population there was a more complex DNA distribution than in the small cells in glioblastomas. These differences were evident using two numerical DNA indices obtained from the analysis of each tumor histogram which defined, respectively, the extent to which the main tumor cell population deviated from euploid values and the probabilities to have multiple stemlines with abnormal DNA content. The results suggest that small anaplastic cells which appear to represent the most aggressive population in glioblastomas have a DNA content diploid-near diploid, and in malignant gliomas the degree of aneuploidy of the main stemline is not related to the biologic behavior of the neoplasm.

Adult↗
PubMed1993

Flow cytometric DNA analysis of gastric cancer--correlation with histology and clinical outcome.

Nuclear DNA content was analysed by means of flow cytometric measurements in 103 patients with gastric carcinomas, using paraffin-embedded archival tissue. DNA aneuploidy was found in 40 cases (38.8%). The mean DNA index of aneuploid tumors was 1.45(range 1.13 to 2.37). No significant association between ploidy and either age, sex, tumor location, size, stage, growth pattern, or histologic type was found. However, the incidence of aneuploidy was higher in high grade carcinomas than in low grade carcinomas; the incidence of aneuploidy was 10%, 68.8%, and 45.8% for Grade II, III, and IV carcinomas, respectively, as compared with Grade I carcinomas which were all diploid. On statistical analysis, Abnormal cellular DNA content was significantly correlated with high histologic grade (P < 0.005). Patients with aneuploid cancer (39.2%) had a poorer prognosis than those with diploid cancer (70.0%) based on (P < 0.01). The 2-year survival rate for advanced gastric carcinoma. Therefore, DNA ploidy might be a useful prognostic factor in cases of advanced gastric cancer.

Adult↗
PubMed1991

[Initial results of image cytometric DNA analysis in the evaluation of prognosis in papillary thyroid cancer].

In this study we analysed DNA-ploidy as a potential prognostic parameter in papillary thyroid carcinoma. Paraffin embedded histological material, obtained by resection from 19 patients with a papillary thyroid carcinoma, was selected for analysis. Tumor areas within the paraffin-embedded material were identified by HE-stained reference sections. One 50 microns section was dewaxed, rehydrated and mechanically and enzymatically prepared to form a suspension of 10,000 cells/ml. 1 ml of the suspension, which contained bare nuclei with small rests of cytoplasma, was centrifuged on glass slides. The fixed nuclei were air-dried and stained by Feulgen SITS technique, which allows for the quantitative measurement of DNA. The DNA analysis was carried out with a computer-controlled single-cell cytophotometry. In contrast to using flow cytometry, only the tumor cells were measured by image-cytometry. Overlapping nuclei, dirt and other artifacts as well as inflammatory cells were efficiently eliminated. With DNA image-cytometry, we could differentiate between diploid (n = 13) and aneuploid (n = 6) tumors. Best prognosis with a survival rate of 92% after 103 months had patients with diploid tumors in contrast to patients with aneuploid tumors who did not survive more than 72 months.

Carcinoma, Papillary↗