Abnormal DNA Content Research Articles

Retinoblastoma cell cycling

Techniques for the measurement of bromodeoxyuridine (BrdUrd) positive cells generally include either microscopic evaluation of paraffin embedded sections or measurements on cell suspensions using a fluorescent activated cell sorter. The accuracy of these measurements and their correlations can be affected by a number of technical and intrinsic tumor factors. Extrinsic parameters including degree of necrosis and tumor growth fraction are less easily analyzed in BrdUrd stained material. Retinoblastoma tumor cell cycling was prospectively studied in 11 children using in vivo and one child using in vitro BrdUrd. BrdUrd measurements were made by staining cell suspensions or sections of paraffin embedded tumor and analyzing by microscopy. Approximately 14% of viable cells were in the synthesis-phase of the cell cycle. The correlation between BrdUrd in cell suspensions and BrdUrd in paraffin embedded sections did not reach significance (r = 0.48). DNA analysis of these tumors was also performed using flow cytometry. Nine tumors were found to have a normal diploid DNA content, one had a G1 peak below the diploid control, two had a G1 peak above the diploid control, and one had two G1 peaks (a diploid and a hyperdiploid peak). There was no correlation between abnormal DNA content and the percent of cells in synthesis.

フローサイトメトリーによる口腔へん平上皮癌核ＤＮＡ量の検討 臨床所見との比較検討

The nuclear DNA contents of 67 oral squamous cell carcinomas were analyzed by flow cytometry and the results were compared with pathological, clinical features and clinical course.All benign tumors had a normal DNA content (diploid pattern) but 44.8% of sluamous cell carcinomas had an abnormal DNA content (ancuploidy pattern). It was found that aneuploidy and DNA Index (DI) were associated with a size of tumor (T), a clinical nodal involvement (N) and clinical stage but not associated with histological grade. The tumors with advanced T, N and stage tended to show an aneuploidy pattern and high DI value. The tumor with pathological nodal involvement had both a high ratio of aneuploid tumors and a high DI value. The occurrence of primary recurrence and distant metastasis might be associated with the rate of aneuploidy. Sevcnty-five percent of 12 dead patients within 3 years had aneuploid tumor (vs. 38% of alive).These findings suggested that flow cytometric analysis might be useful to predict the prognosis of oral s: luamous cell carcinomas.

Cellular DNA Content and Proliferative Activity Evaluated by Flow Cytometry versus Histopathological and Staging Classifications in Human Bladder Tumors

Flow cytometric analysis of cellular DNA content was performed on 78 biopsy bladder samples obtained from 61 patients with bladder tumors. All 6 normal tissue samples and 1 benign papilloma exhibited a cytometrically diploid DNA distribution, while 39 of 60 bladder carcinomas exhibited at least one aneuploid cell subpopulation. Furthermore, 13 of 39 aneuploid tumors were characterized by the presence of more than one aneuploid cell subpopulation. The results indicate a significant relationship between cytometric ploidy and morphological classification and stage: the occurrence of subpopulations with abnormal DNA content is associated with the increase in differentiation grade and stage. A significant statistical difference in the survival pattern between the diploid and aneuploid groups was observed. The percent of S cells extracted from DNA content distribution histograms indicates a statistically significant difference (p less than 0.01) between normal tissue (3.7 +/- 1.8), diploid tumor (8.4 +/- 3.9) and aneuploid tumor (14.9 +/- 6.0). Moreover the percent of S-phase cells increases with grade in only the aneuploid subgroup. Our results suggest that cytometric parameters in association with morphological and clinical criteria can contribute to a more accurate characterization of bladder tumors in prognostic terms.

DNA content prognostic in soft tissue sarcoma: 102 patients followed for 1–10 years

In a prospective study of 102 patients with soft tissue sarcoma, the prognostic importance of DNA content and clinicopathologic features was analyzed. Based on DNA flow cytometry, 37 lesions were diploid (normal DNA content) and 65 were nondiploid (abnormal DNA content). The 5-year metastasis-free survival rate of the whole series was 0.59. The survival rate was 0.77 for patients with diploid tumors and 0.48 for those with nondiploid tumors (P = 0.01). Multivariate analysis identified two independent metastatic risk factors: increasing tumor size and nondiploidy. Unexpectedly, high malignancy grade (III-IV) was not found to be an independent risk factor for metastasis. The risk of metastasis was strongly related to the number of risk factors present. Thus, the 5-year survival for the 76 patients with no or one risk factor was 0.69, as compared with 0.30 for the 26 patients with two risk factors (P less than 0.0001). Our study shows that metastatic disease in soft tissue sarcoma is closely related to nondiploidy. A prognostication model based on DNA content and tumor size was found to discriminate between patients with a good and a poor prognosis after surgical treatment. The model can be used to identify patients who should be excluded from trials with adjuvant chemotherapy.

Recognition of the CDEl motif GTCACATG by mouse nuclear proteins and interference with the early development of the mouse embryo

We have reported previously (1) two unexpected consequences of the microinjection into fertilized mouse eggs of a recombinant plasmid designated p12B1, carrying a 343 bp insert of non-repetitive mouse DNA. Injected at very low concentrations, this plasmid could be established as an extrachromosomal genetic element. When injected in greater concentration, an early arrest of embryonic development resulted. In the present work, we have studied this toxic effect in more detail by microinjecting short synthetic oligonucleotides with sequences from the mouse insert. Lethality was associated with the nucleotide sequence GTCACATG, identical with the CDEl element of yeast centromeres. Development of injected embryos was arrested between the one-cell and the early morula stages, with abnormal structures and DNA contents. Electrophoretic mobility shift and DNAse foot-printing assays demonstrated the binding of mouse nuclear protein(s) to the CDEl-like box. Base changes within the CDEl sequence prevented both the toxic effects in embryos and the formation of protein complex in vitro, suggesting that protein binding at such sites in chromosomal DNA plays an important role in early development.

DNA analysis of multiple synchronous renal cell carcinomas

The authors used retrospective quantitative DNA analysis to study interrelationships between multiple synchronous renal cell carcinomas in seven patients. DNA content was determined by image analysis on Feulgen-stained nuclear smears prepared from multiple paraffin blocks from each tumor. Tumors were unilateral in four cases and bilateral in three. Ten tumors had homogeneous, and four heterogeneous DNA stemlines. Intertumoral heterogeneity in four cases suggested multifocal origin. Identical DNA aneuploid indices in bilateral tumors in one case suggested metastasis from a solitary origin. Abnormal DNA content and heterogeneous populations began to appear in the size range 2.0 to 5.0 cm. All tumors over 5.0 cm contained nondiploid populations. Although the interrelationships between these multiple synchronous neoplasms are not entirely clear, the DNA analysis suggests that the occurrence of nondiploid stemlines and heterogeneous DNA content may parallel both tumor growth and more aggressive behavior.

The influence of tumour cell DNA content on survival in colorectal cancer: a detailed analysis

We have investigated the influence of tumour cell DNA content (ploidy) on survival of 416 patients undergoing excisional surgery for colorectal cancer. Two hundred and eleven (51%) tumours had an abnormal DNA content (aneuploid or tetraploid). There was no correlation between ploidy status, sex, age and pathological stage, histological grade, tumour site, local tumour extension or assessment of curability. Patients with tumours with an abnormal DNA content had a poorer survival 68/211 (32%) than patients with near normal (diploid) DNA content 88/205 (43%) (test statistic 5.0, P = 0.02). The patient subgroups in which DNA content exerted an influence on survival were: stage B tumours (P = 0.0058), moderately differentiated tumours (P = 0.004), rectal tumours (P = 0.02), and mobile tumours (P = 0.02). Multivariant analysis showed that pathological stage, local tumour extension and DNA ploidy were all independent prognostic indicators whereas histological grade, tumour site and assessment of 'curability' were not. The influence of pathological stage, however, was much greater than that of local tumor extension or DNA ploidy. Tumour cell DNA content together with pathological stage and local tumour extension may be used in a prognostic index and may be important in planning adjuvant therapy.

Flow Cytometric DNA Content of Adenoid Cystic Carcinoma of Submandibular Gland: Correlation of Histologic Features and Prognosis

Flow cytometric analysis of nuclear DNA content was performed in 26 adenoid cystic carcinomas of the submandibular gland using archived, paraffin-embedded tissues. The DNA content was compared with multiple histologic parameters and clinical course. Ten carcinomas (38%) were aneuploid and 16 (62%) diploid. Aneuploid carcinomas demonstrated a higher frequency of solid cytoarchitecture, lymph node metastases, and advanced clinical stage, as compared with diploid carcinomas. Other histologic features predicting aggressive clinical behavior also correlated with abnormal DNA content and included invasion of nerves larger than 0.25 mm and intravascular extension. Our data suggest that DNA content analysis can be an effective objective parameter in the clinicopathologic assessment of adenoid cystic carcinoma.

A clinical and flow cytometric analysis of patients with nasopharyngeal cancer

Abnormal cellular DNA content, a hallmark of malignancy, is known to be an important prognostic factor in many human solid tumors; however, no data have been published on whether cellular DNA content carries prognostic significance for patients with nasopharyngeal cancer (NPC). Archival, formalin-fixed, paraffin-embedded pathology specimens representing pretreatment tissue biopsies from 55 patients (41 men and 14 women) with NPC were analyzed for cellular DNA content in a retrospective fashion from 1968 to 1988. Individual tumors were classified as either lymphoepithelioma, squamous cell, or anaplastic carcinoma, and were staged according to International Union Against Cancer (UICC) criteria. All patients were treated with curative intent using a 4 to 6 MeV linear accelerator to total doses ranging from 50 to 60 Gy in 4 to 6 weeks. The overall 5-year actuarial survival for all 55 patients was 44.4% (men, 41%; women, 52%). Survival by T stage was as follows: T1, 65%; T2, 51%; T3, 36%; and T4, 27%. Similarly, the 5-year survival rate declined as the bulk of nodal metastases increased: N0, 62%; N2, 50%; N3, 37%; and N1, 25%. Patients who had anaplastic carcinoma had a 5-year survival of 73%, those with lymphoepithelioma had a 60% survival, and those with squamous cell cancer (SCC) had a 30% survival. There was a statistically significant difference in 5-year survival between patients with SCC and those with nonkeratinizing histologies (P less than 0.05). In addition, there was a significant association between patients older than 40 years of age with SCC and patients younger than 40 years of age with nonkeratinizing malignancies (P less than 0.01). Of the 55 tumors successfully analyzed, 22 (40%) were diploid and 33 (60%) were aneuploid. The mean coefficient of variation (CV) of all 55 samples was 6.17%. There was no significant difference in 5-year survival between patients with diploid and those with aneuploid tumors (48% versus 42%). Furthermore, there was no statistically significant survival difference between aneuploid and diploid tumors within any one histologic subgroup. There was also no significant survival difference related to the DNA index. The results indicate that the extent of local tumor spread is still the most important prognostic factor for patients treated with radiotherapy for NPC. The data support the conclusion that patients with lymphoepithelioma and anaplastic carcinomas have a superior survival to patients with squamous cell carcinoma.(ABSTRACT TRUNCATED AT 400 WORDS)

DNA measurements in chronic hepatitis, cirrhosis and hepatocellular carcinoma

It has been documented that chronic hepatitis may progress to cirrhosis and then develop hepatocellular carcinoma (HCC). To test whether abnormal cellular DNA increases along this line of development, liver tissues from 48 patients with chronic hepatitis, 17 with cirrhosis, and 8 with HCC were investigated for cellular DNA content with a scanning microdensitometer. Seven of 8 HCCs and 2 cirrhotic livers adjacent to HCC had abnormally increased cellular DNA content. Only 4 livers from patients with chronic liver diseases other than HCC had abnormal cellular DNA content. The cellular DNA content in livers not accompanying HCC was not related to the patient's age, histological diagnosis, and hepatitis inflammatory activity. The results confirmed the increase of cellular DNA content in HCC, but did not provide evidence of a progressively increasing DNA content from chronic hepatitis to liver cirrhosis. However, cirrhotic livers with abnormal hepatocytic DNA content deserve careful follow-up for the early detection of HCC.

DNA quantitation and histologic characteristics of squamous cell carcinoma of the upper aerodigestive tract : W Sakr, M Hussan, RJ Zarbo, et al. Arch Pathol Lab Med 1989; 113:1009–1014

DNA analysis is currently being applied to many solid tumors to determine DNA content and synthesis phase fraction. We compared DNA content measured by flow cytometry with multiple histologic parameters in 155 squamous cell carcinomas of the upper aerodigestive tract. Abnormal DNA content (aneuploidy) was identified in 107 (69%) of the neoplasms. Abnormal DNA content or aneuploidy correlated with above-median synthesis phase fraction and increased frequency of mitotic figures. Other histologic features predicting aggressive tumor behavior also correlated with abnormal DNA content and included small cords or single cell pattern of tumor invasion, high nuclear grade, and decreased stromal or desmoplastic response to the invading squamous cell carcinoma.

Flow cytometric DNA analysis of thyroid carcinoma

Abnormal DNA content has been considered as an additional criterion for determining the biological behavior of a tumor. Flowcytometric DNA analysis was done on 121 patients with thyroid carcinoma encountered during the period between 1975 and 1987. Tumor tissues were sampled from paraffin-embedded blocks and the histology of thyroid carcinoma found to consist of 91 papillary, 23 follicular, 2 medullary, 1 squamous cell and 4 anaplastic carcinomas. The incidence of aneuploidy in thyroid carcinoma was 7.4 per cent (9 patients) while that of diploidy was 92.6 per cent (112 patients). The aneuploid specimens consisted of 6 papillary, 1 follicular, 1 medullary and 1 anaplastic carcinomas and, of 4 anaplastic carcinoma patients with subsequent death within 6 months, only 1 was aneuploid. As an indicator of proliferative potential, S-phase fraction (SPF) was also determined by flow cytometry, but this could not be used as an independent prognostic factor. The aneuploid patients showed a significantly decreased survival rate (p less than 0.01). Thus, although DNA measurement proved useful for predicting the survival of aneuploid patients, there is some discrepancy between DNA content and the biological behavior of the tumor.

Flow cytometric DNA ploidy analysis of squamous cell carcinoma of the oral cavity: Comparison with clinical staging and histologic grading

The clonal DNA content of tumor biopsy specimens of 110 patients with primary and untreated squamous cell carcinoma of the oral cavity was determined by flow cytometric study. The ploidy status was compared with tumor size and histologic grading. Thirty tumors (27.3%) were diploid; in 80 cases (72.7%) cell lines with abnormal DNA content were detected. The portion of aneuploid tumors increased with decreasing degree of histologic differentiation (P less than 0.001) from G1 (38.1%) to G2 (76.6%) and G3 (92.0%). Only one of 13 T1 carcinomas (7.6%) showed abnormal clonal DNA content, but 76.9% of T2 and 90.6% T3 tumors (P less than 0.001) did. The emergence of aneuploid clones obviously represents a marker of malignancy progression in oral carcinoma.

Stromal neoplasms of the breast: A comparative flow cytometric study

We analyzed 42 mammary spindle cell neoplasms (10 conventional fibroadenomas, 8 giant fibroadenomas, 17 cystosarcoma phyllodes, and 7 stromal sarcomas) by flow cytometry to assess the diagnostic and prognostic relevance of DNA content analysis in the pathologic evaluation of these lesions. Our data indicate that all fibroadenomas were diploid, cystosarcoma phyllodes displayed diploid and aneuploid DNA content irrespective of their histological categorization, and that stromal sarcomas were all aneuploid and clinically aggressive. Diploid cystosarcoma phyllodes were biologically indolent, whereas most of the aneuploid neoplasms killed their hosts (P = 0.03). The present study indicates that a DNA content abnormality it is a reliable marker of malignancy in these neoplasms, and it may identify subsets of patients with variable biologic courses within the histopathologic spectrum of cystosarcoma phyllodes.

DNA Ploidy in Pancreatic Neuroendocrine Tumors

The nuclear DNA content of 17 pancreatic neuroendocrine tumors was measured from paraffin-embedded tissue with flow cytometry. The tumors were classified by immunostaining with antisera for synaptophysin, insulin, gastrin, glucagon, pancreatic polypeptide, somatostatin, and vasoactive intestinal polypeptide. Eight (47%) of the 17 tumors were aneuploid, and two (12%) were multiploid (had two aneuploid stemlines of cells). Seven of the eight insulinomas, one of the four gastrinomas, and two of the four nonspecified neuroendocrine tumors had an abnormal nuclear DNA content. The DNA indices of the aneuploid and multiploid cases ranged from 1.13 to 1.93, and three cases had a DNA index greater than 1.50. During the follow-up for up to 16 years (mean, 7 years), one patient with diploid nonspecified tumor died of the disease, another patient with a multiploid gastrinoma had metastatic disease develop, and a third patient with a multiploid nonspecified tumor was alive with the disease. The authors conclude that many neuroendocrine tumors of the pancreas have an abnormal nuclear DNA content as measured by DNA flow cytometry. DNA multiploid pancreatic neuroendocrine tumors may be associated with a less favorable clinical course, but this needs to be confirmed in additional studies.

DNA flow cytometry of acinic cell carcinomas of major salivary glands

Fifteen acinic cell carcinomas from an equal number of patients were analysed for their DNA content and proliferative (S-phase) index by flow cytometry from archival tissues. Seven of the carcinomas manifested a diploid DNA content. None of the patients with diploid acinic cell carcinomas died of their carcinomas and none developed metastases in follow-up periods extending for 10 or more years. Four of eight patients with aneuploid acinic cell carcinomas have died because of their malignancies within a 10 year period after the first surgical removal of the carcinoma. Five of the eight patients exhibited metastases. Although the number of cases does not permit strong correlations between histopathological features, abnormalities in DNA content and outcome of patients, it was noted that carcinomas with prominent necrosis, tubuloductal differentiation and 'dedifferentiated' areas displayed more aggressive biological courses.

Tissue carcinoembryonic antigen and dna aneuploidy in precancerous and cancerous colorectal lesions

Chronic inflammatory bowel disease (CIBD) and colorectal adenoma are considered as precancerous conditions and lesions of large bowel carcinoma, respectively. They, therefore, may be used to study the behavior of such different factors as tumor-associated antigens and nuclear DNA content abnormalities in colorectal carcinogenesis. Tissue concentrations of carcinoembryonic antigen (CEA) were significantly higher in those precancerous lesions (CIBD: 61 +/- 11.2 ng/mg, adenoma: 70 +/- 6 ng/mg; mean +/- standard error of the mean) than in normal colonic mucosa (36 +/- 4.7 ng/mg). Colorectal carcinoma had still higher tissue levels (437 +/- 108.2 ng/mg). No correlation between tissue CEA and tumor differentiation could be found, but there was a significant difference between aneuploid (747 +/- 354 ng/mg) and diploid (139 +/- 43 ng/mg) tumors. Using flow cytometry DNA aneuploidy was present in 31.6%, 10.5%, and 51.6% of CIBD, colorectal adenoma, and carcinoma, respectively. These data suggest that the occurrence of aneuploidy is not strongly dependent on a malignant transformation, but it may also be present in premalignant colorectal lesions without cellular dysplasia.

Carotid Body Paragangliomas: A Clinicopathologic and DNA Analysis of 13 Tumors

The clinical and pathological features of 13 carotid body paragangliomas from 12 patients were examined and correlated with the DNA ploidy pattern as determined by image analysis. These tumors occurred in 7 women and 5 men aged 19 to 62 years (average, 42 years). All presented with a slowly enlarging, usually asymptomatic mass of 2 weeks' to 25 years' duration. Two patients were related and had a family history of paragangliomas. The tumors ranged from 2 to 6 cm. All contained scattered chief cells with pleomorphic nuclei, two exhibited mitoses, and three showed perineural and three vascular invasion. Follow-up was available in all 12 patients and ranged from 15 months to 28 years (average, 7.3 years). None of the tumors recurred locally, but one did metastasize to a single cervical lymph node that was apparent at the time of diagnosis. Of 13 carotid body paragangliomas examined for DNA, 4 were diploid, 3 diploid-tetraploid, 3 tetraploid, 2 aneuploid, and 1 polyploid. The only malignant tumor was polyploid. From these observations, we conclude that abnormalities in DNA content of carotid body paragangliomas are common and that tumor ploidy cannot be used to assess malignant potential. We also found no apparent relationship among nuclear pleomorphism, mitotic activity, perineural invasion, or vascular invasion and clinical behavior. Perineural and vascular invasion, however, were observed only in tumors with abnormal DNA histograms.

DNA Content of Juvenile Granulosa Tumors Determined by Flow Cytometry

Juvenile granulosa tumors (JGT) often exhibit worrisome morphologic features, yet usually behave in a benign fashion. Thirteen JGT were examined by flow cytometric analysis of paraffin material to determine if DNA content and cell kinetics are related to prognosis. The patients ranged in age from stillborn to 16 years. Unilateral salpingoophorectomy was the most common therapy. Eleven patients with follow-up were free of disease. Marked nuclear atypia was evident in three cases, and high mitotic counts were observed in six, but only marked atypia correlated with DNA content. Flow cytometry revealed that 46% of the JGT had abnormal DNA content and increased average growth fraction. The neoplasms with the highest DNA indices were found predominantly in postmenarchal girls. JGT may exhibit abnormal DNA content, nuclear atypia, and numerous mitoses, yet behave benignly. DNA flow cytometric studies of higher stage JGT are warranted.

Significance of DNA content abnormalities in small rectal cancers

Several studies have shown that the presence of DNA content abnormalities, measured by flow cytometry, may correlate with a poor prognosis in a variety of cancers. The predictive value of DNA content in patients with small rectal cancers has not been well determined. Thirty-nine patients with primary rectal adenocarcinoma smaller than 3 cm were studied in a comparison of DNA content with established prognostic variables. The following covariates were evaluated for their prognostic value: sex, age, tumor size, location, distal margin, Dukes' classification, tumor differentiation, and DNA content. DNA content was assessed by flow cytometric analysis, and each tumor was categorized as diploid or nondiploid. Of the parameters studied, Dukes' classification and tumor DNA content were found to be independent prognostic indicators. Determination of DNA content seems to provide additional useful prognostic information in patients with small rectal tumors.