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PubMed1992

Flow cytometric DNA analysis of ovarian Brenner tumors and transitional cell carcinomas.

Flow cytometry has been previously used as a method of obtaining prognostic information about ovarian carcinomas using ploidy, DNA index, and S-phase fraction. DNA content has also been assessed in ovarian tumors of low malignant potential. Brenner tumor variants such as metaplastic, proliferating, and low malignant potential, recently designated as intermediate Brenner tumors, and malignant Brenner tumors are unusual tumors that present classification problems. Their histological appearance may not accurately reflect biological activity. We used flow cytometry to analyze paraffin-embedded tissue for DNA content and S-phase in 34 Brenner tumors, three ovarian transitional cell (urothelial) carcinomas (TCCs), and nine normal control ovaries. We correlated histological and clinical features with DNA analysis. Twenty-five Brenner tumors and three ovarian TCCs were acceptable for histogram analysis (coefficient of variation less than 7.0). Thirteen typical, three metaplastic (extensive mucinous or glandular metaplasia), and two proliferating (papillary formation with increased cellularity) Brenner tumors were diploid. One proliferating tumor was tetraploid. The single Brenner tumor of low malignant potential was diploid but had an increased S-phase. Four of five malignant Brenner tumors were aneuploid, and one was diploid. All the TCCs were aneuploid. S-phase was elevated in intermediate and malignant Brenner tumors and TCC. Limited numbers of cases available preclude prognostic prediction based on ploidy in malignant Brenner tumors or primary ovarian TCCs. DNA ploidy and S-phase reflect the intermediate status of metaplastic, proliferating, and low malignant potential Brenner tumors.

Adult↗
PubMed1995

Flow cytometric DNA analysis of extramammary Paget's disease of the vulva.

Vulvar extramammary Paget's disease (EMPD) is an uncommon disease entity that occurs predominantly in postmenopausal white women. The clinical behavior of this neoplasm is extremely variable, reflecting the various histological patterns that have been reported with this lesion. Flow cytometry has been used as a method of obtaining prognostic information about a number of gynecological neoplasms, yet to date there have been no flow cytometric studies performed on this unusual neoplasm. We performed flow cytometric analysis of 14 cases of paraffin-embedded vulvar EMPD in patients ranging from 45 to 86 years of age. We correlated histological features and clinical recurrence risk with DNA analysis. Although we were unable to show a statistically significant correlation between DNA ploidy or S-phase and time to recurrence, we did show a statistically significant correlation between DNA ploidy and histological features. Aneuploidy appears to be associated with in situ sweat gland adenocarcinoma, invasive carcinoma, and lymphatic invasion. These results suggest that Paget's cells with aneuploid DNA stem lines may be associated with the potential for aggressive biologic behavior.

Aged↗
PubMed1985

Prenatal diagnosis of classic phenylketonuria by DNA analysis.

Prenatal diagnosis of classic phenylketonuria (PKU) was performed in two at-risk families by means of a cloned human phenylalanine hydroxylase gene probe which was used to analyse DNA isolated from cultured amniotic fluid cells. The diagnoses of a PKU fetus in one family and a heterozygous fetus in another family were confirmed after birth. The prenatal diagnosis procedure by DNA analysis can be confidently applied to 90% of caucasian families with previously affected children.

DNA↗
PubMed1990

Mitochondrial DNA analysis of Exophiala jeanselmei and Exophiala dermatitidis.

Mitochondrial DNA (mtDNA) analysis with Hae III, Hind III and Msp I was performed in 45 Exophiala jeanselmei strains (30 Phialophora jeanselmei and 154 Phialophora gougerotii strains) and 31 Exophiala dermatitidis strains. The results were as follows, 1) P. jeanselmei and P. gougerotii are identical, 2) E. jeanselmei is classified into 18 types based on restriction profiles, 3) two strains of E. jeanselmei CBS 577.76 and CBS 578.76 are identified as E. dermatitidis, 4) E. dermatitidis has no intraspecific variation and is definitely distinct from E. jeanselmei, 5) E. jeanselmei is suggested to be a complex organism because of extensive mtDNA polymorphism.

DNA, Fungal↗
PubMed2004

[Random amplified polymorphic DNA analysis of the genomes among 7 species of ticks].

OBJECTIVE: To study genomic polymorphic DNA and genetic distance of 7 species of ticks. METHODS: Ticks used in this study were Dermacentor nuttalli, D. silvarum, Haemaphysalis qinghaiensis, H. formosensis, H. punctata, Amblyomma testudinarium, and Ixodes ovatus. DNA extracts of the 7 species of ticks were amplified by random amplified polymorphic DNA (RAPD) and PCR technique using 5 primers with different arbitrary single chain polynucleotide sequences. DNA fingerprint maps were analyzed and the genetic distance among 7 species of ticks were counted. RESULTS: The amplified products of the 7 species of ticks by RAPD all showed their specific DNA band. The average genetic distance among them was 0.71. CONCLUSION: RAPD can differentiate the 7 species of ticks.

Animals↗
PubMed1990

Microphotometric nuclear DNA analysis in cervical dysplasia of the uterine cervix: its relation to the progression to malignancy and regression to normalcy.

The present study was attempted to reveal the predictive value of DNA ploidy pattern of uterine cervical dysplasia cases in relation to cervical carcinogenesis. Microphotometric nuclear DNA analysis using Feulgen stain was carried out in 310 cervical smears of 80 dysplasia cases consisting of 53 cases which progressed to malignancy and 27 cases which regressed to inflammation or normalcy during their follow-up periods. Aneuploid DNA pattern was observed in initial as well as follow-up smears in 69.8% of cases of the progressive group, and in 7.4% of cases of the regressive group. This difference is statistically highly significant (chi 2 27.88, p less than 0.001). In the progressive group, an aneuploid DNA value was observed in 40.0% of mild dysplasia, 71.9% of moderate dysplasia and 90.9% of severe dysplasia. In the regressive group, DNA aneuploidy was observed in only 9.1% of moderate dysplasia. This difference is statistically significant. These findings indicate that an aneuploid DNA value is a risk indicator for malignant potential of dysplasia cases.

Adult↗
PubMed1991

Flow cytometric DNA analysis of hepatocellular carcinoma.

The prognostic value of nuclear DNA content was studied retrospectively using flow cytometry in 203 cases of resected hepatocellular carcinoma. The occurrence of DNA aneuploidy, which was detected in 50% of patients, correlated significantly with tumor size and the presence of vascular invasion or intrahepatic metastasis. Overall, patients with DNA aneuploid tumors had a significantly worse prognosis than those with DNA diploid tumors (P less than 0.001) and, also in subdivided groups by tumor size (P less than 0.01). Among DNA aneuploid patients, the survival times were significantly shorter for patients with a low DNA index (less than 1.5) than for those with a high DNA index (greater than or equal to 1.5) (P less than 0.05). In a Cox multivariate analysis, nuclear DNA content provided significant prognostic value (P = 0.008), as did vascular invasion (P = 0.001) and intrahepatic metastasis (P = 0.005). These results indicated that nuclear DNA content has an important prognostic value in hepatocellular carcinoma.

Carcinoma, Hepatocellular↗
PubMed2006

Mitochondrial DNA analysis of ancient Peruvian highlanders.

Ancient DNA recovered from 57 individuals excavated by Hiram Bingham at the rural communities of Paucarcancha, Patallacta, and Huata near the famed Inca royal estate and ritual site of Machu Picchu was analyzed by polymerase chain reaction, and the results were compared with ancient and modern DNA from various Central Andean areas to test their hypothesized indigenous highland origins. The control and coding regions of the mitochondrial DNA (mtDNA) of 35 individuals in this group were sequenced, and the haplogroups of each individual were determined. The frequency data for the haplogroups of these samples show clear proximity to those of modern Quechua and Aymara populations in the Peruvian and Bolivian highlands, and contrast with those of pre-Hispanic individuals of the north coast of Peru that we defined previously. Our study suggests a strong genetic affinity between sampled late pre-Hispanic individuals and modern Andean highlanders. A previous analysis of the Machu Picchu osteological collection suggests that the residents there were a mixed group of natives from various coastal and highland regions relocated by the Inca state for varied purposes. Overall, our study indicates that the sampled individuals from Paucarcancha and Patallacta were indigenous highlanders who provided supportive roles for nearby Machu Picchu.

DNA, Mitochondrial↗
PubMed1987

Prognostic value of computerized DNA analysis in noninvasive papillary carcinomas of the urinary bladder.

DNA parameters and a DNA status index were calculated in cases of noninvasive urothelial papillary carcinomas and apparently normal urothelium. DNA feature analysis included the measurement of basic parameters as well as those related to DNA deviation. Normal urothelium had a diploid content with a few values between 2c and 4c. The papillary lesions in the three grades showed a progressive decrease in diploid nuclei and steadily increasing percentages of tetraploid ones. One case of grade 1 papillary carcinoma and some of grade 2 showed a small proportion of aneuploid cells; the proportion quintupled in grade 3. The values of parameters related to DNA deviation increased progressively in the three grades, numerically expressing the shift from diploidy. The DNA status index is a multiparameter classification in which a single number, ranging from 0.5 to 3.7, expresses the degree of DNA deviation from a non-proliferating diploid population. The cases of normal urothelium and of grade 1 had the lowest values, from 0.5 to 1.9, whereas grade 3 cases had the highest, from 2.2 to 3.7. The index values of grade 2 cases were partly intermediate and partly in the range of the neighboring grades. The DNA status index has a prognostic significance. In fact, associated lesions and recurrences were only observed in cases with index values over 2.0.

Carcinoma, Papillary↗
PubMed1997

Sample preparation from paraffin-embedded tissue specimens for laser scanning cytometric DNA analysis.

We have developed a simple, rapid method for isolating cells from a block of formalin-fixed, paraffin-embedded tissue specimen for laser scanning cytometric (LSC) DNA analysis by using a grater. The scraping-like tissue samples were obtained by grating a paraffin-embedded tissue block. The grated samples were collected, put into a small plastic tube, and deparaffinized with xylene. Subsequently, the samples were immersed in 100% ethanol to remove the xylene. After using a syringe with a 26-gauge needle and filtering through 40-microm nylon mesh, the cells suspended in ethanol were dropped directly onto a glass slide. As a result, isolated cells adhered tightly to the glass slide. The slides mounted with isolated cells were treated with 0.1% pepsin in 0.1 N HCl for 1 h at 37 degrees C and then 0.1% RNase for 10 min at room temperature. The slides were dipped in propidium iodide (25 microg/ml) to stain DNA and sealed with nail varnish. The coefficients of variation for histograms were small enough to detect an aneuploid peak close to the diploid peak.

Aneuploidy↗
PubMed1994

Papillary-cystic tumor of the pancreas in a young woman: fine-needle aspiration cytology, ultrastructure and DNA analysis.

A case of papillary-cystic tumor (PCT) of the pancreas in a young woman is reported. Fine-needle aspiration (FNA) was done preoperatively under ultrasound guidance. The aspirate showed numerous delicate papillary fragments, dyscohesive and monomorphic tumor cells with folded nuclear membranes, and foamy macrophages. A diagnosis of PCT of the pancreas was made based on clinical, radiologic, and cytologic findings. The patient underwent distal pancreatectomy without complications. The histopathologic examination of the surgical tissue confirmed the diagnosis of PCT of the pancreas. The tumor cells were faintly positive with mucicarmine and periodic acid-schiff (PAS) stains. Immunocytochemistry using Ki67 monoclonal antibody showed a cycling index of 0.1 percent, supporting the clinical observation of low metastatic and recurrence rates of this rare tumor. DNA analysis of the tumor showed a DNA index of 1.09 (diploid) and an S-phase fraction of 5.38%. The tumor cells were positive for progesterone receptors (> 15 fmol/mg protein) but negative for estrogen receptors (< 15 fmol/mg protein). Abundant mitochondria, prominent endoplasmic reticulum and few junctional complexes were noted on electron microscopy. Emphasis is placed on accurate diagnosis based on preoperative FNA cytology in order to maximize cure rates while minimizing surgical risk and complications.

Adult↗
PubMed1996

The role of flow cytometric DNA analysis in determining prognosis of resectable ductal adenocarcinoma of the pancreas.

Carcinoma of the pancreas is a leading cause of cancer mortality in the United States. Improvement in prediction of survival is needed. Flow cytometric analysis as a prognostic tool has produced conflicting results. We retrospectively analyzed the clinicopathologic features, operative factors, and outcome of 39 curative resections for ductal adenocarcinoma of the head of the pancreas performed at Indiana University Medical Center between 1989 and 1994. The group was composed of 20 females and 19 males. Procedures performed were Whipple without vagotomy (n = 5), Whipple with vagotomy (n = 19), pylorus-preserving Whipple (n = 12) and total pancreatectomy (n = 3). Thirty-two tumors were suitable for DNA analysis. Of the 32 patients with flow cytometric data, 33 per cent (3/9) of living patients and 39 per cent (9/23) of deceased patients had aneuploid tumors (P = 0.999). The average S-phase for living patients was 8.3 per cent +/- 3.8 per cent, and 16.1 per cent +/- 13.6 per cent for deceased patients (P = 0.115). In the multivariate analysis, only lymphatic invasion (P = 0.015) and alkaline phosphatase level (P = 0.024) predicted poor survival. Our data show no correlation between flow cytometric DNA ploidy, S-phase analysis, and prognosis in patients undergoing curative resection for ductal adenocarcinoma of the pancreatic head.

Adenocarcinoma↗
PubMed1988

Flow cytometric DNA analysis of malignant fibrous histiocytoma and related fibrohistiocytic tumors.

Fibrohistiocytic neoplasms with similar histologic characteristics may have vastly different biologic behaviors. We studied 23 fibrohistiocytic tumors to determine if cellular DNA content was correlated with clinical outcome. Archival paraffin blocks of 9 malignant fibrous histiocytomas (MFH), 3 dermatofibrosarcoma protuberans, 9 dermatofibromas, 1 juvenile xanthogranuloma, and 1 nodular fasciitis were processed, stained with propidium iodide, and analyzed by flow cytometry. Five of 9 (56%) MFH and 1 of 3 (33%) dermatofibrosarcoma protuberans were aneuploid. All 11 benign fibrohistiocytic tumors were diploid. Local recurrence occurred in 3 of 5 (60%) cases of aneuploid MFH, but in none with a diploid MFH tumor. No cases of dermatofibrosarcoma protuberans or benign tumors recurred. Aneuploidy was associated with decreased survival, as 2 of 5 patients with aneuploid MFH died within 1 year of diagnosis whereas all 4 patients with diploid MFH tumors are alive after an average follow-up of 4 years (range, 1 to 11). The diploid and aneuploid groups did not differ in clinical stage at the time of diagnosis. Our results indicate that retrospective DNA analysis can detect aneuploidy in both MFH as well as other fibrohistiocytic tumors, and that aneuploidy in MFH may place the patient at increased risk for local recurrence and mortality.

DNA↗
PubMed2004

Human genomic DNA analysis using a semi-automated sample preparation, amplification, and electrophoresis separation platform.

The growing importance of analyzing the human genome to detect hereditary and infectious diseases associated with specific DNA sequences has motivated us to develop automated devices to integrate sample preparation, real-time PCR, and microchannel electrophoresis (MCE). In this report, we present results from an optimized compact system capable of processing a raw sample of blood, extracting the DNA, and performing a multiplexed PCR reaction. Finally, an innovative electrophoretic separation was performed on the post-PCR products using a unique MCE system. The sample preparation system extracted and lysed white blood cells (WBC) from whole blood, producing DNA of sufficient quantity and quality for a polymerase chain reaction (PCR). Separation of multiple amplicons was achieved in a microfabricated channel 30 microm x 100 microm in cross section and 85 mm in length filled with a replaceable methyl cellulose matrix operated under denaturing conditions at 50 degrees C. By incorporating fluorescent-labeled primers in the PCR, the amplicons were identified by a two-color (multiplexed) fluorescence detection system. Two base-pair resolution of single-stranded DNA (PCR products) was achieved. We believe that this integrated system provides a unique solution for DNA analysis.

Automation↗
PubMed2005

[DNA-analysis in hereditary cancer: the importance of a reliable family history].

Three patients, a 45-year-old man, a 51-year-old woman and a 43-year-old woman, wanted to know whether they had a hereditary predisposition for cancer. The family of patient A fulfilled the clinical diagnostic criteria for hereditary non-polyposis colorectal carcinoma (HNPCC). The family of patient B fulfilled the clinical diagnostic criteria for hereditary breast/ovarian cancer (HBOC). The family of patient C did not completely fulfil the criteria for HBOC since only two family members had a confirmed diagnosis of breast cancer. In all three families, DNA-mutation analysis was performed. In families A and B no mutation was found. However, based on the family history, the diagnosis of hereditary cancer was made and recommendations for surveillance were given. After extensive counselling, one member of family B eventually decided to have prophylactic surgery performed. A few years later, a pathogenic mutation in BRCA2 was found in family B. In family C, an unclassified variant was found in BRCA1. Further investigations in the family were not possible, due to a lack of co-operation from family members. It is important to obtain a thorough and complete family history. When DNA-analysis remains inconclusive or if an unclassified variant is found, recommendations for surveillance will be based on this family history.

Adult↗
PubMed1989

Use of low-frequency-cleavage restriction endonucleases for DNA analysis in epidemiological investigations of nosocomial bacterial infections.

Epidemiological investigations of bacterial infections are generally based on multiple phenotypic markers that are often difficult to verify. A more general and reliable method is genomic DNA analysis by restriction endonucleases. However, the commonly used endonucleases produce too many fragments for correct separation by agarose electrophoresis. In contrast, simple electrophoretic patterns are obtained after genomic DNA digestion by low-frequency-cleavage restriction endonucleases and pulsed-field gel electrophoresis, making it easier to compare numerous strains from the same species. This technique was used to investigate an Acinetobacter calcoaceticus outbreak in a urologic department and bronchial colonization of artificially ventilated patients by Pseudomonas aeruginosa in an intensive care unit. The method allowed a clear distinction between epidemic and self-contaminating strains in these different epidemiological situations.

Acinetobacter↗
PubMed1999

Parotid gland lesions: diagnosis of malignancy with MRI and flow cytometric DNA analysis and cytology in fine-needle aspiration biopsy.

BACKGROUND: The purpose of this study was to assess the capability of magnetic resonance imaging (MRI) and cytology and flow cytometric (FCM) deoxyribonucleic acid (DNA) analysis in fine-needle aspiration biopsy (FNAB)-derived materials for diagnosing malignancy of the parotid lesions and the efficacy of FCM analysis in FNAB. METHODS: Magnetic resonance imaging findings and FCM results (ploidy and S + G2 + M phases [S + G2M] fraction) and cytology in FNAB-derived materials in 26 patients with 26 parotid lesions (12 benign lesions, 14 malignancies) were assessed for predicting malignancy. Flow cytometric results in aspirates were compared with those in surgically resected tissues. RESULTS: When a single predictor was used, cytology (92% accuracy) was most accurate for malignancy, followed by ill-defined margin (88% accuracy) and aneuploidy (88% accuracy). The combination of FCM and cytology raised the rate of sufficient materials from 92% to 100% and accuracy from 92% to 96% compared with cytology alone. The same highest accuracy (96%) was obtained with the combination of the ill-defined margin or other findings such as cytology, aneuploidy, or a high (S + G2M) fraction (6% <). Deoxyribonucleic acid ploidy in the FNAB showed full agreement with that in the surgical specimens. Receiver operating characteristic curves showed that the diagnosis of malignancy with (S + G2M) fraction in FNAB was superior to that in surgical specimens, but no significant difference was noted. CONCLUSIONS: A combination of MRI findings, cytology, and FCM results is optimal for diagnosing malignancies of the parotid lesions, and FNAB may replace the surgical specimens in FCM analysis.

Adolescent↗