Value Of DNA Content Research Articles

Prognostic value of DNA index, S‐phase fraction and p53 protein accumulation after surgical resection of esophageal squamous‐cell carcinomas in Thailand

The prognostic value of cell nuclear DNA content, S-phase fraction and p53 protein accumulation in esophageal squamous-cell carcinomas was studied in a consecutive series of 80 patients from a high-incidence region of southern Thailand, who underwent esophagectomy between 1983 and 1993. Flow cytometry was used to determine tumor ploidy, DNA index and S-phase fraction, while p53 protein accumulation was evaluated immunohistochemically using the monoclonal anti-p53 antibody, CO7. Biomarkers were correlated with clinico-pathologic findings and survival by univariate and multivariate analysis. p53 protein was found in 40 tumors (50%), and was associated with significantly reduced overall survival. In patients with immunopositive tumors, depth of primary tumor invasion, lymph-node status. TNM stage and tumor grade were also significant prognostic factors. Additional predictors of reduced overall survival after esophagectomy, determined by flow cytometry, included S-phase fraction above 10%, aneuploidy (DNA index 1.2-1.8) and multiploidy (DNA index > 2.2). This study further implicates p53 in the pathogenesis of esophageal squamous-cell carcinoma. Prognostic factors such as p53 protein, S-phase fraction and DNA index may be useful in stratifying patients for adjuvant therapies in future clinical trials of esophageal cancer.

208 G2 block in irradiated cervix tumors

Uterine cervix carcinoma is one of the most common gynaecological malignancies in Portugal, accounting for 19.6% of all tumors in women. Considerable interpatient differences in radioresponse are observed in this condition, and thus it is important to evaluate the role that tumor radiosensitivity may play in determining treatment efficacy. The demonstration that some tumors are more radiosensitive than others may allow the selection of a subgroup that will need adjuvant therapy. The objective of this work was to verify the predictive value of DNA content and G2 phase fraction, evaluated by flow cytometry, on the outcome of fractionated radiotherapy. A significant radiation induced in G2 block was observed after one week of treatment, both in aneuploid and diploid tumors.

Nuclear DNA content and nucleolar organizer regions in colorectal cancer.

The prognostic value of nuclear DNA content and argyrophilic nucleolar organizer regions (AgNOR) is still controversial in colorectal cancer. Sixty patients with colorectal cancer were studied by flow cytometric DNA analysis and AgNOR measurement, and their prognostic significance was tested. DNA index was closely linked to depth of invasion and lymph node metastasis, while AgNOR count did not correlate with such parameters. The survival curve was strongly influenced by depth of invasion, lymph node metastasis, and Dukes' stage but was not affected by DNA ploidy and AgNOR count. These results indicate that neither DNA ploidy nor AgNOR count correlates with survival of patients, although DNA ploidy is linked to progression of colorectal cancer.

Porcine Hepatocytes From Slaughterhouse Organs

Most recent strategies for the development of hybrid artificial liver devices focus on the use of parenchymal liver cells (hepatocytes). For clinical application of these devices, a sufficient cell supply is mandatory. Because human liver tissue is rarely available, isolated porcine hepatocytes from laboratory animals have been suggested for use in bioartificial livers. The authors introduce a modified isolation protocol to yield large scale numbers of viable porcine hepatocytes from slaughterhouse organs. Perfusion and enzymatic digestion of the left medial liver lobe (n = 74) resulted in 1.0 +/- 0.3 x 10(7) viable hepatocytes per gram of tissue, and an overall yield of 1.92 +/- 0.5 x 10(9) viable cells per isolation (viability: 93 +/- 2%). Collagen gel immobilization maintained morphologic integrity and functional activity of hepatocyte cultures over long-term periods. Cell morphology, as assessed by light microscopic evaluation, was maintained for 2 weeks. Stable DNA content (51 +/- 5 micrograms) and low values of alanine aminotransferase release (8 microU/hr/micrograms DNA) indicated structural stability of cultures after a short period of post isolational adaptation. Albumin secretion (4.5 micrograms/hr/micrograms DNA) and persistent Cytochrome P450 IA1 dependent deethylation of 7-ethoxycoumarin (4.5 nmol/hr/micrograms DNA) indicated long-term metabolic activity of cultured hepatocytes. Hepatocytes from livers of slaughtered pigs represent an unlimited resource of viable material for cell culture, and their usefulness as functional units of bioartificial liver support devices should be tested.

The prognostic value of DNA content in colorectal cancer

The prognostic value of DNA content in colorectal cancer

Prognostic value and clinicopathologic correlation of p53 gene mutations and nuclear DNA content in human lung cancer: A prospective study: Casson AG, McCuaig S, Craig I, Ayed A, Inculet R, Kerkvliet N et al. Mount Sinai Hospital, 600 University Ave., Toronto, Ont. M5G 1X5. J Surg Oncol 1994;56:13–20

Prognostic value and clinicopathologic correlation of p53 gene mutations and nuclear DNA content in human lung cancer: A prospective study: Casson AG, McCuaig S, Craig I, Ayed A, Inculet R, Kerkvliet N et al. Mount Sinai Hospital, 600 University Ave., Toronto, Ont. M5G 1X5. J Surg Oncol 1994;56:13–20

LACK OF DIAGNOSTIC-VALUE OF DNA CONTENT AND P53 IMMUNOSTAINING IN NORMAL, HYPERPLASTIC AND NEOPLASTIC PARATHYROID TISSUE

The aim of this project was to study the diagnostic value of DNA content and p53 protein expression in normal, hyperplastic and neoplastic parathyroid lesions. Tissue samples of 74 parathyroid glands from 34 patients with primary hyperparathyroidism were studied by DNA flow cytometry and p53 immunostaining. In 9 of 23 patients (39%) with parathyroid adenoma, a nondiploid cell population was present. Some normal looking glands removed from the same patients also had a nondiploid DNA index. Multiglandular hyperplasia was found in 11 patients, and in 5 of these (45%) the histograms showed nondiploid cells. The proliferative activity was generally low and S-phase fraction did not differ in glands with hyperplasia or adenoma, when compared with normal looking glands. One single case of hyperplasia showed a weak p53 positivity in scattered nuclei, probably representing wild type p53 protein. Thus, our present results suggest that DNA content and p53 protein staining are of no value in the routine work up of parathyroid glands removed from patients with primary hyperparathyroidism.

DNA Content as Prognostic Factor in Cervix Carcinoma Stage IB-III Treated with Radiotherapy

A prognostic value of DNA content in cervix carcinoma for local recurrence following radiotherapy is suggested by both retro- and prospective studies. The purpose of this study was to confirm these data. Flow cytometry and computerized image cytometry were used to measure DNA content of tumor cells retrospectively in paraffin-embedded archive material from 98 patients with carcinoma of the uterine cervix FIGO stage IB-III. All patients were treated with combined megavoltage irradiation and brachytherapy. Minimum follow-up has been 5.1 years. Pelvic recurrence was seen in 10, 26, and 54% of patients with stages IB, II, and III, respectively ( P = 0.003). DNA content, expressed as DNA index (DI), was not correlated with the FIGO stage ( P = 0.25), histopathologic grading ( P = 0.26), or age ( P = 0.63) at diagnosis in this series. Using univariate analysis, pelvic disease-free survival (PDFS) was best predicted by the clinical stage ( P = 0.004) and by an age greater than 55 years ( P = 0.04) before treatment. PDFS tended to be better for patients with aneuploid tumors (DI > 1.2), but this difference was not statistically significant ( P = 0.17). Using Cox's regression model for multivariate analysis, only stage was independently correlated to PDFS ( P = 0.009). After stratification for stage, ploidy level was significantly correlated with PDFS in stage II patients ( P = 0.04). Overall results suggest aneuploid tumors to be more radiosensitive than diploid tumors in this series.

Prognostic value of deoxyribonucleic acid content in prostate cancer: a review of current results.

A total of 115 articles on prostate cancer were reviewed for data on the prognostic value of DNA content in the tumor cells. In 44 series, data pertinent to this review were found. There was no consensus in the literature with respect to methods of analysis of DNA content or definitions of subclasses of DNA content such as categories of ploidy. The DNA content of prostate cancer cells was strongly related to tumor grade and stage. When analyzed as a single parameter in univariate analyses, the DNA content had a prognostic value with respect to overall or disease-specific survival. In multivariate analyses the additional prognostic value of the DNA content was less convincing when analysed with tumor grade and stage. The prognostic data from univariate and multivariate analyses available in the literature were mainly derived from patients with advanced disease and data on localized, potentially curable disease were scanty and conflicting.

Prognostic value and clinicopathologic correlation of p53 gene mutations and nuclear DNA content in human lung cancer: a prospective study.

The aim of this prospective study was to determine whether use of a combination of biomarkers, p53 and nuclear DNA content, led to improved prognosis and clinicopathologic correlation in human non-small cell lung cancer. Nineteen patients undergoing curative resection of primary non-small cell lung cancer were evaluated. Resected tumors were studied by polymerase chain reaction/single strand conformation polymorphism analysis (p53 gene mutations), flow cytometry (nuclear DNA content and cell cycle analysis), and immunohistochemically (p53 oncoprotein). Histologically normal lung was used as an internal control for each patient. Minimum postoperative follow-up was 4 years. p53 gene mutations (5/19 tumors; 26%), tumor ploidy (5/19 diploid), patterns of immunoreactivity, or combination of biomarkers did not appear to correlate with clinicopathologic findings or clinical outcome. Two of three patients with associated second primary malignancies, had squamous cell diploid tumors with p53 gene mutations. We conclude that p53 gene mutations and tumor ploidy may represent different biologic markers for human non-small cell lung cancer. Although trends in improved predictive accuracy were not seen when both markers were incorporated into the tumor analysis, flow cytometry and molecular analysis of the p53 gene may identify patients at increased risk of the development of a second primary malignancy.

Nuclear DNA content of commercially important Eucalyptus species and hybrids

This paper reports the nuclear DNA content estimates obtained by flow cytometry for a group of twelve Eucalyptus species and five fast-growing hybrids that includes those most widely planted throughout the world. Estimates of nuclear (2C) DNA content for the species surveyed ranged from 0.77 pg/2C for Eucalyptuscitriodora Hook. (subgenus Corymbia) to 1.47 pg/2C for Eucalyptussaligna Smith (subgenus Symphyomyrtus). This range corresponds to a haploid genome size range of 370–700 megabase pairs. The average physical equivalent of a 1 cM distance could be as low as 200 kilobase pairs in Eucalyptus, an attractive feature for positional cloning efforts in woody plants. The closer the species were in phylogenetic relationship the more similar were their nuclear DNA content values. All the interspecific hybrids surveyed displayed a nuclear DNA content in the expected intermediate range between the respective parental species, with the exception of one originating from Rio Claro, Brazil, whose exact parentage is unknown. No evidence of polyploidy was observed in any of the hybrids. The flow cytometry procedure employed in this study is an efficient method for investigating ploidy levels of high yielding hybrids of Eucalyptus.

Cellular survival and proliferation in autogenous flexor tendon grafts

In order to investigate fibroblast survival and proliferation in autogenous flexor tendon grafts, hindlimb intrasynovial and extrasynovial donor tendons were placed within the synovial sheaths of the medial and lateral forepaw digits of 21 dogs (42 tendons) and treated with controlled early passive motion. Intravital histologic evaluations with confocal microscopy and biochemical determinations of total DNA content and DNA synthesis were carried out at 10 days, 3 weeks, and 6 weeks. Intravital staining of the extrasynovial tendon grafts demonstrated variable degrees of cellular necrosis at the earliest intervals followed by cellular repopulation with fibroblasts and neovascularization from surface vessels. In contrast, intrasynovial tendon grafts were populated predomnantly by viable cells at each interval, with occasional patches of cell necrosis and fibroblast ingrowth. Total DNA content and DNA synthesis values in the intrasynovial donor tendons were significantly lower than those seen in the extrasynovial tendon grafts at each interval. Extrasynovial tendons appear to act as scaffolds, undergoing extensive cellular death followed by a rapid repair response. Findings that intrasynovial tendon fibroblasts survive the tendon grafting process suggest that the nutritional supplies and metabolic requirements of intrasynovial and extrasynovial donor tendons differ largely.

Tumor-cell DNA content predicts outcome in children and adolescents with clinical group III embryonal rhabdomyosarcoma. The Intergroup Rhabdomyosarcoma Study Committee of the Children's Cancer Group and the Pediatric Oncology Group.

The prognostic value of tumor-cell DNA content (ploidy) was evaluated in children with unresectable, nonmetastic rhabdomyosarcoma of embryonal histology. Flow-cytometric techniques were used to estimate the ploidy of tumor specimens from 34 patients with embryonal rhabdomyosarcoma who were enrolled in the intergroup rhabdomyosarcoma study III (IRS III) from 1985 to 1991. Tumors were classified as diploid or hyperdiploid (DNA content, 1.1 to 1.8 times that of normal diploid cells). The influence of ploidy on clinical outcome was assessed by the Kaplan-Meier technique and Cox regression analysis with stepwise selection. Twelve of the tumor specimens were diploid and 22 were hyperdiploid. The patient groups defined by diploid or hyperdiploid tumors had similar presenting characteristics (eg, age, tumor size, and anatomic site). Significantly more children with hyperdiploid tumors achieved a complete response than did children with diploid tumors (85% v 42%; P = .01). The probability of progression-free survival at 5 years (+/- SE) was 91% +/- 6% for the hyperdiploid group, compared with 17% +/- 11% for the diploid group (P < .001). Hyperdiploidy was also associated with a significantly higher overall survival rate at 5 years: 96% +/- 4% versus 50% +/- 14% (P = .004). Ploidy retained its prognostic significance after adjustment for tumor site in the Cox regression model. Tumor-cell ploidy strongly correlates with outcome in children with nonmetastic, unresectable embryonal rhabdomyosarcoma. The two biologically distinct groups identified by this measure would benefit from further refinements in risk-directed therapy.

On the Question of the Cause of the Initial Slope of Survival Curves

In the double-strand break (DSB) model, the initial slope (alpha) of the survival curve is determined by the number of DSBs remaining unrepaired at a certain time after irradiation. There are three types of calculations which confirm the validity of this approach. First, the model quite reasonably predicts the low-LET alpha values for Chinese hamster V79, mouse L5178Y-R, normal fibroblast, ataxia telangiectasia (AT), and Ehrlich ascites tumor (EAT) cells using the measured values of the cellular DNA content, the radiation yield for DSB induction, the time constant for DSB repair, and the time available for DSB repair. Second, this approach correctly predicts an increase in alpha values for V79 and Chinese hamster ovary (CHO) cells due to postirradiation treatments with hypertonic saline or a high concentration of araA. These calculations are based on the duration of the DSB repair block induced by those treatments. Third, a fair agreement between the predicted and measured initial slopes for 3- to 4-MeV alpha particles was obtained for normal fibroblast, EAT, V79, CHO, Chinese hamster HS-23, C3H 10T1/2, and lung epithelial cells. However, the alpha values for AT and xrs-6 cells were overestimated. The calculation of high-LET alpha values uses the experimental values of the relative increase in the DSB repair time constant and in the radiation yield for DSB induction for alpha particles in comparison with those for X rays.

Assessment of the cellular DNA content of whole mounted mouse and human oocytes and of blastomeres containing single or multiple nuclei.

The nuclear DNA content of intact, live or fixed, human and mouse oocytes and blastomeres has been measured rapidly and reliably. Chromosomal DNA has been stained with DAPI, the fluorescent emission from which has been measured photocytometrically. In vitro fertilised mouse oocytes and embryos at various stages of development were assessed for their DNA content. The mean values of 1C, 2C and 4C DNA content were clearly different, and it was possible to assign correctly individual values for DNA content to each class with 92%, 61% and 81% confidence respectively. Maintaining the cells as whole mounts allowed other morphological and structural features to be examined. When formation of multiple micronuclei was induced in mouse oocytes by their insemination in the presence of nocodazole, the additive signal from all the micronuclei in one zygote was equivalent to the expected DNA content. Application to early human blastomeres of this photocytometric technique for measurement of the total cellular DNA content revealed that multinucleated blastomeres contained 2C to 4C DNA levels, consistent with a diploid DNA content.

Prognostic Value of Cellular DNA Content and S‐Phase Fraction in Head and Neck Squamous Cell Carcinomas in Relation to Different Treatment Modalities

AbstractDNA‐ploidy and the percentage of S‐phase cells were measured by DNA flow cytometry in 171 primary squamous cell carcinomas of the head and neck. Of the analyzed tumors, 102 (60%) were aneuploid and 69 (40%) were diploid. Minimum follow‐up was 6 months (median: 37 months). For the whole population no difference of the 5‐year survival rate was found comparing diploid (18%) and aneuploid tumors (26%). Following sequential radiochemotherapy, aneuploid tumors revealed a better 5‐year survival (54%) than diploid tumors (7%; P &lt; 0.01). Accordingly, the locoregional control rate was higher for aneuploid tumors (57% vs. 28%; n.s.). By contrast, following surgery and radiotherapy or concurrent radiochemotherapy, diploid and aneuploid tumors had nearly the same survival rates. The analysis of recurrence demonstrated a lower rate of locoregional failures of diploid tumors (surgery + radiotherapy: 32% vs. 44%), but this was leveled out by a higher incidence of distant metastases of diploid carcinomas (surgery + radiotherapy: 36% vs. 17%). Relating to the fraction of S‐phase cells, slowly proliferating tumors (S‐phase &lt; median; 28%) had a better 5‐year survival rate than fast proliferating tumors (20%; P &lt; 0.05). The same was true for different treatment modalities surgery + radiotherapy; concurrent or sequential radiochemotherapy) and for the investigated stage III and IV tumors according to UICC 1987. The analysis of recurrence revealed that the better survival of slowly proliferating tumors was explained by the higher locoregional control rate (60%) compared to fast proliferating tumors (45%; P &lt; 0.05). Stepwise multivariate analysis revealed treatment modality (P = 0.107), S‐phase fraction (P = 0.026), and UICC stage (P = 0.044) as independent prognostic factors for locoregional recurrences and S‐phase fraction (P = 0.0143) for 3‐year overall survival. These data indicate that flow cytometrically evaluated DNA‐ploidy and S‐phase fraction have a relevant prognostic power for predicting the clinical outcome of squamous head and neck cancer. © 1993 Wiley‐Liss, Inc.

Predictive value of DNA content in head and neck carcinoma (HNC) treated with hyperfrationated radiotherapy (HRT)

Predictive value of DNA content in head and neck carcinoma (HNC) treated with hyperfrationated radiotherapy (HRT)

The prognostic value of DNA content measured by image cytometry in soft tissue sarcomas.

Nuclear DNA content in soft tissue sarcoma was determined by image cytometry using archival, paraffin embedded material. In a retrospective study 138 specimens of 81 patients have been analysed. The ploidy level was correlated to clinical outcome regarding tumor volume and histological grading, the most important prognostic parameters. Ploidy has a significant prognostic value and correlates well with histological grading (p = 0.01). Tumour volume was found to be an independent prognostic factor (p = greater than 0.1) [chi 2 test]. The DNA content of the primary tumour and of multiple local recurrences remained similar.

Correlation of DNA ploidy, histopathology, stage and clinical outcome in gastric carcinoma

The determination of nuclear DNA ploidy from paraffin-embedded specimens was performed by flow cytophotometry on 277 surgically resected primary gastric carcinomas to assess the relationship of various pathological findings and DNA content with survival. The preparation of samples was performed by a modification of Hedley's technique and the staining method of Vindelov. Eighty-nine (32%) carcinomas were DNA diploid, 69 (25%) were DNA tetraploid, and 119 (43%) were DNA aneuploid. DNA non-diploid patterns were significantly associated with macroscopic ulcerative appearance, location of the tumour in the proximal stomach, histological grade, and advanced stage of tumour. Patients with DNA non-diploid cancers, and specifically DNA aneuploid cancers, exhibited significantly poorer survival than patients with DNA diploid tumours. These data support the prognostic value of tumour DNA content in patients with resected gastric carcinoma.

Measurement of Bromodeoxyuridine Labeling Index, Ki-67 Score and Ag- NOR Count in Breast Carcinomas

Nuclear DNA content and three cell proliferation markers, the bromodeoxyuridine (BrdUrd) labeling index, Ki-67 score and Ag-nucleolar organizer region (Ag-NOR) counts, were measured in 21 cases of breast carcinoma, and then relationships among them were investigated. The BrdUrd labeling indices and Ki-67 scores were strongly correlated with one another (r = 0.90, p less than 0.001). They were also slightly correlated with the Ag-NOR counts (p less than 0.05). Although growth activity varied greatly from case to case, even within the same histologic grade in breast carcinoma, high histologic grade carcinomas were frequently associated with high values of both the BrdUrd labeling index and Ki-67 score, as compared with low histologic grade ones. The DNA ploidy level (DNA index) was independent of these cell proliferation markers. In the present series, 17 of 21 cases (81%) were aneuploid. All cases without DNA aneuploidy fell into a group of low histologic grade, while 7 of 11 low grade tumors were aneuploid. All three cell proliferation markers were low in 2 of the 4 cases without DNA aneuploidy. It may be possible to identify cases with favorable prognosis in a group of low grade carcinomas on the grounds of diploid DNA content and low values of these markers.