DNA Diploidy Research Articles

A novel clinical tool and risk stratification system for predicting the event-free survival of neuroblastoma patients: A TARGET-based study.

Neuroblastoma (NB), considered the most common non-intracranial solid tumor in children, accounts for nearly 8% of pediatric malignancies. This study aimed to develop a simple and practical nomogram to predict event-free survival (EFS) in NB patients and establish a new risk stratification system. In this study, 763 patients primarily diagnosed with NB in the Therapeutically Applicable Research to Generate Effective Treatments (TARGET) database were included and randomly assigned to a training set (70%) and a validation set (30%) in a 7:3 ratio. First, the independent prognostic factors of EFS for NB patients were identified through univariate and multivariate Cox regression analyses. Second, a nomogram was created based on these factors and was validated for calibration capability, discriminative, and clinical significance by C-curves, receiver operating characteristic (ROC) curves, and decision curve analysis. Finally, a new risk stratification system was established for NB patients based on the nomogram. The univariate Cox analysis demonstrated that NB patients with age at diagnosis >318 days, International Neuroblastoma Staging System (INSS) stage 4, DNA diploidy, MYCN amplification status, and children oncology group (COG) high-risk group had a relatively poor prognosis. However, according to the multivariate Cox regression analysis, only age, INSS stage, and DNA ploidy were independent predictive factors in NB patients regarding EFS, and a nomogram was created based on these factors. The area under the curve (AUC) values of the ROC curves for the 3-, 5-, and 10-year EFS of this nomogram were 0.681, 0.706, and 0.720, respectively. Additionally, the AUC values of individual independent prognostic factors of EFS were lower than those of the nomogram, suggesting that the developed nomogram had a higher predictive reliability for prognosis. In addition, a new risk stratification system was developed to better stratify NB patients and provide clinical practitioners with a better reference for clinical decision-making. NB patients' EFS could be predicted more accurately and easily through the constructed nomogram and event-occurrence risk stratification system, allowing clinicians to better differentiate NB patients and establish individualized treatment plans to maximize patient benefits.

Meta-analysis of the predictive value of DNA aneuploidy in malignant transformation of oral potentially malignant disorders.

DNA aneuploidy is an imbalance of chromosomal DNA content that has been highlighted as a predictor of biological behavior and risk of malignant transformation. To date, DNA aneuploidy in oral potentially malignant diseases (OPMD) has been shown to correlate strongly with severe dysplasia and high-risk lesions that appeared non-dysplastic can be identified by ploidy analysis. Nevertheless, the prognostic value of DNA aneuploidy in predicting malignant transformation of OPMD remains to be validated. The aim of this meta-analysis was to assess the role of DNA aneuploidy in predicting malignant transformation in OPMD. The questions addressed were (i) Is DNA aneuploidy a useful marker to predict malignant transformation in OPMD? (ii) Is DNA diploidy a useful negative marker of malignant transformation in OPMD? These questions were addressed using the PECO method. Five studies assessing aneuploidy as a risk marker of malignant change were pooled into the meta-analysis. Aneuploidy was found to be associated with a 3.12-fold increased risk to progress into cancer (RR=3.12, 95% CI 1.86-5.24). Based on the five studies meta-analyzed, "no malignant progression" was more likely to occur in DNA diploid OPMD by 82% when compared to aneuploidy (RR=0.18, 95% CI 0.08-0.41). In conclusion, aneuploidy is a useful marker of malignant transformation in OPMD, although a diploid result should be interpreted with caution.

The value of cell cycle analysis by propidium-iodine staining of CD56+ cells in pediatric brain tumors

PurposeCell cycle analysis by flow cytometry has not been adequately studied in pediatric brain tumors. We investigated the value of cell cycle analysis by propidium-iodine (PI) staining of CD56+ (gated) cells for the characterization of pediatric brain tumor's malignancy. MethodsPatients that underwent surgery for an intracranial lesion and tissue sample was available were included in the study. All tumor samples were histopathologically verified before flow cytometric analysis. ResultsThere were 46 pediatric brain tumor cases. As control we used samples from three cases of brain tissue obtained during surgery for epilepsy. All tumors had significant lower G0/G1 and significant higher S-phase, G2/M fraction, S+G2/M and S+G2/M/G0/G1-phase fraction than normal brain tissue. Low-grade tumors had significant lower S-phase than high grade tumors. Grade IV tumors had significant lower G0/G1 fraction and higher S+G2/M/G0/G1 index than grade III tumors. DNA diploidy was detected in 24 tumors and DNA aneuploidy was detected in 23 tumors. There was a significant correlation between Ki-67 index and both S+G2/M and S+G2/M/G0/G1 phase fraction. ConclusionsCell cycle analysis by PI staining of CD56+ cells is a promising method for the assessment of pediatric brain tumors malignancy and could be a valuable adjunct to histopathological evaluation.

DNA Aneuploidy in Porcine Bone Marrow–Derived Mesenchymal Stem Cells Undergoing Osteogenic and AdipogenicIn VitroDifferentiation

In this study, we estimated the distribution of DNA diploidy and aneuploidy in porcine mesenchymal stem cells (pMSCs) that were subjected to osteoblast/osteocyte and adipocyte differentiation to determine the impact of long-term in vitro culture and differentiation on the cell cycle distribution and nuclear DNA profile. This determination could be helpful to confirm or exclude the suitability of physico-chemical culture conditions for the purposes of both the maintenance of an undifferentiated state and to promote differentiation in pMSCs. Flow cytometry was applied to analyze the cell cycle and occurrence of aneuploidy/diploidy, and real-time PCR was used to quantify aP2 and osteocalcin, markers of adipocytes and osteocytes, respectively. The chi-squared test was used to compare the total rates of G0/G1-, S-, and G2/M-phase cell fractions with diploid and aneuploid DNA and the DNA index ratios between three experimental groups of pMSCs. Five weeks of in vitro culture under differentiating conditions resulted in a considerable reduction of DNA stability and a remarkable increase in the rate of cells exhibiting an aneuploid DNA stem line; however, a similar dependence was not found in the nondifferentiated MSCs. Furthermore, the cell fraction rates in each phase of the mitotic cycle and the DNA index (DI) were calculated. The results of real-time PCR for aP2 and osteocalcin proved positive MSC differentiation toward adipocytes and osteocytes. In terms of the possible use of differentiated MSC lines in tissue engineering and regenerative medicine, we propose cytokinetic diagnostics using flow cytometry as an objective and useful method for screening the tumor-forming capacity and malignancy potential of both in vitro long-term cultured MSCs and MSCs subjected to ectopic differentiation.

Effects of DNA Ploidy and S-Phase Fraction on Fluorine-18 FDG Uptake of Primary Breast Cancer Lesions

BackgroundFlow cytometry (FCM) evaluating DNA content is emerging as the tool to monitor cell proliferation and malignant potential in several cancers such as stomach, lung, and salivary gland tumor. The purpose of this study was to correlate 18F-FDG uptake of dual–time-point (DTP) positron emission tomography (PET) imaging with DNA ploidy and S-phase fraction (SPF) in primary breast cancer lesions. Patients and MethodsSeventy-two consecutive female patients (mean age ± SD, 52.7 ± 11.1 years; range, 28-81 years) had undergone 18F-FDG DTP PET/computed tomography (CT) imaging for staging of breast malignancy in our institution during a 5-month period. FCM was performed on fresh-frozen samples of specimens obtained from surgery. 18F-FDG uptake was then compared with DNA content. ResultsForty-four malignant lesions were included in this study. On FCM, DNA aneuploidy was detected in 14 lesions (31.8%). The maximum standardized uptake values (SUVmax1 and SUVmax2) (SUVmax1, 6.8 ± 4.6 vs. 3.4 ± 2.4; P = .017), (SUVmax2, 7.9 ± 5.7 vs. 3.6 ± 2.7; P = .015) and retention index (RI) (12.8 ± 11.6 vs. 2.4 ± 10.8; P = .010) were significantly higher in DNA aneuploidy cancer than in DNA diploidy cancer. The value of RI (11.3 ± 11.5 vs. 2.6 ± 11.2; P = .022) was significantly higher in high SPF (> 15%) breast cancer than in low SPF (≤ 15%) breast cancer. ConclusionsHigh 18F-FDG uptake in breast cancer might be an indicator of DNA aneuploidy and high SPF. We propose that 18F-FDG PET/CT imaging may be a noninvasive and useful tool for predicting the DNA content in breast cancer.

Ploidy and S-phase fraction as predictive markers of response to radiotherapy in cervical cancer

The aim of this study was to investigate the potential clinical utility of DNA flow cytometry biomarkers, ploidy, and S-phase fraction (SPF) in predicting overall survival in cervical cancer. This prospective study involved 159 patients with cervical carcinoma (median follow-up, 48 months). Pretreatment clinical staging was done according to the FIGO 2009 update classification. Biopsy tumor samples were used for flow cytometry analysis and histological examination. A prognostic study was performed using both Cox and Bayesian Weibull regression models. Eighty (50.3%) tumors presented DNA aneuploidy, mostly observed in adenosquamous (AS) cell carcinoma (8 of 9 cases) and adenocarcinoma (AC) (12 of 17 cases). The median SPF value (8.6%) was used for discriminating low vs. high tumor cell proliferation. High SPF significantly correlated with aneuploidy ( p < 0.001). All AS carcinomas had SPF > 15%, while all ACs presented SPF < 10% ( p < 0.001). Forty-three (27%) patients died of the disease during follow-up. Log-rank tests revealed significant differences between survival curves for older patients (≥44 years) ( p = 0.029), advanced clinical staging ( p < 0.001), and DNA diploidy in stage IIB of disease ( p = 0.039). Both regression analyses showed that advanced clinical staging and low SPF independently predict worse overall survival of patients. The results suggest that DNA flow cytometry parameters can provide additional predictive information in cervical cancer management.

Positive expression of c-myc and p53 products in two cases of pulmonary sclerosing hemangioma

The c-myc and p53 genes are thought to be an oncogene and a tumor suppressor gene, respectively. These genes' products are characteristic of malignant tumors. We quantitatively analyzed the c-myc and p53 products by flow cytometry in two cases of pulmonary sclerosing hemangioma. In case 1, 32.3% of the tumor cells were found to have the c-myc product, and 8.9% were found to have the p53 product. In case 2, 6.7% of the tumor cells were found to have the c-myc product and 15.5% were found to have the p53 product. The percentages in both cases were twice as high as those in a negative control lymphocytes stained with c-myc and p53 products. Therefore, these two cases showed positive expression of the c-myc and p53 products. In addition DNA from six other patients with sclerosing hemangioma was analyzed with paraffin-embedded sections. All six had DNA diploidy, with DNA indexes ranging from 0.91 to 1.03 and coefficients of variation ranging from 3.0 to 5.5. We suggest that pulmonary sclerosing hemangioma is a very weakly malignant tumor.

Detection of intratumoral DNA heterogeneity in primary lung cancer using a multiple sampling method

The nuclear DNA contents of 18 freshly resected specimens of primary lung cancer tissue were determined by flow cytometry with a multiple sampling method. A DNA aneuploidy pattern was observed in all cases. In three cases, a diploid DNA pattern was observed along with abnormal DNA stem lines. Twelve cases (66%) had two or more abnormal DNA stem lines, and were classified as DNA multiploidy. The frequencies of DNA aneuploidy and DNA multiploidy were higher than in previously reported studies. Intratumoral DNA heterogeneity was observed in 10 cases (55%). Three of these were a mixture of DNA diploidy and DNA aneuploidy, and the other seven were a mixture of DNA aneuploidy and DNA multiploidy. If multiple sampling had not been performed in these cases, their ploidy patterns would have been misinterpreted. We conclude that multiple sampling is important in determining the precise DNA ploidy patterns of primary lung cancers.

Pancreatitis‐Associated Protein Is Related Closely to Neoplastic Proliferative Activity in Patients with Colorectal Carcinoma

Pancreatitis-associated protein (PAP) is a secretory protein that is not only expressed during acute pancreatitis but also in pancreatic adenocarcinoma, gastric carcinoma, hepatocellular carcinoma, and colorectal carcinoma. Expression in carcinoma might be another characteristic of PAP. The aim of our study was to assess, in 27 patients undergoing surgery for colorectal carcinoma, the expression of the PAP mRNA and to evaluate its association with DNA ploidy and proliferative activity (S-phase fraction, SPF) by reverse transcriptase-polymerase chain reaction (RT-PCR) and flow cytometric analysis (FCM). PAP mRNA was expressed in 29.6% (8 of 27) of the patients with colorectal carcinoma. DNA aneuploid and high SPF were found in 87.5% (7 of 8) of patients with PAP mRNA positive colorectal carcinoma. The serum PAP level was significantly elevated in patients with colorectal carcinoma when compared with the healthy subjects. Twelve of the 27 patients with colorectal carcinoma had high serum PAP concentrations (>25 ng/mL) and the mean SPF was 17.82% +/- 8.02%, which was significantly higher compared with the normal colorectal tissue group (7.33% +/- 3.18%, P < 0.05). The mean serum PAP concentration of DNA aneuploidy colorectal carcinomas was 46.67 +/- 17.58 ng/mL, which was significantly different when compared with the DNA diploidy group (19.18 +/- 8.89 ng/mL, P < 0.05). PAP mRNA expression and serum PAP levels are closely related to neoplastic proliferative activity in patients with colorectal carcinoma. No significant differences are observed between PAP mRNA expression and clinicopathologic parameters (P > 0.05).

Prospective study on prognostic significance of DNA ploidy and Ki-67 expression in colorectal cancer

21184 Background: The prognostic value of flow cytometry DNA ploidy and Ki-67 expression in colorectal carcinomas has not been defined yet. The study tries to correlate tumoral DNA ploidy and proliferative activity (Ki-67) with therapy response, Overall Survival (OS), Disesase Specific Survival (DSS) and Disease Free Survival (DFS). Methods: From 01/09/02 to 30/06/05, 3 samples of cancer tissue and 1 sample of normal mucosa has been collected from all operating pieces of colorectal cancer. These samples were frosen and later disgregated, treated and coloured with Propidium Iodide. DNA ploidy was evaluated by FACSCalibur cytofluorimetry. Normal mucosa tissue was our internal control. Ki-67 was evaluated by immuno-histochemistry in all tumoral samples. All the patients were cured with chemo- and/or radiotherapy in our divisions. Results: 67 patients (M/F 35/32); median age was 70; staging: 19% I, 33% II, 30% III, 18% IV. We found aneuploidy in 65,7% of carcinoma and Ki-67 median expression was 55%. DNA tumoral heterogeneity was present in 27% of patients. DNA aneuploidy correlates with advanced disease stage at diagnosis (p&lt;0,01), with high number of metastatic lymphnodes (p&lt;0,005) and with serological markers positivity (p&lt;0,04). After surgery and chemotherapy 35% of the patients with aneuploid carcinoma and high proliferative activity (Ki-67&gt;55%) do not show evident disease versus 100% of patients with DNA diploidy and lower Ki-67. Tumoral aneuploidy correlates in a significative way with lower OS (48% vs 89% of diploid patients), lower DSS (tumor death happened just in patients with aneuploid DNA in every disease stages), with lower DFS (18% vs 86% of diploid patients). Univariated analysis stated that aneuploidy determinates an Odd Ratio=5,7 to develop disease progression (p=0,033). At the moment Ki-67 expression with 55% cut-off does not seem to correlate with OS, DSS and DFS. Conclusions: Preliminar results (the study is still in progress) seem to suggest that cytofluorimetric DNA-ploidy has a prognostic and predictive significance in colo-rectal carcinomas. Ki-67 expression (immuno-histochemistry) has an uncertain significance. The small number of patients and the short follow-up do not allow us to reach any definitive conclusions, but the study is worth to go on. No significant financial relationships to disclose.

Headliners: Reproductive Health: Sperm DNA Changes as Men Age

Vol. 114, No. 9 EnvironewsOpen AccessHeadliners: Reproductive Health: Sperm DNA Changes as Men Age Jerry Phelps Jerry Phelps Search for more papers by this author Published:1 September 2006https://doi.org/10.1289/ehp.114-a526AboutSectionsPDF ToolsDownload CitationsTrack Citations ShareShare onFacebookTwitterLinked InReddit Wyrobek AJ, Eskenazi B, Young S, Arnheim N, Tiemann-Boege I, Jabs EW, et al. 2006. Advancing age has differential effects on DNA damage, chromatin integrity, gene mutations, and aneuploidies in sperm. Proc Natl Acad Sci U S A 103:9601–9606.In the past several decades, more men and women have been postponing parenthood. Fatherhood among men aged 35–49 has increased 40%, while there has been a 20% decline in births fathered by men under age 30. Although it has long been accepted that women face reproductive challenges with age, the consequences of delaying fatherhood have been less understood. Now NIEHS grantees Andrew J. Wyrobek of Lawrence Livermore National Laboratory and Brenda Eskenazi of the University of California, Berkeley, School of Public Health, with their colleagues, have produced new research that suggests that, like women, men too have a “biological clock,” but one that causes a more gradual change in fertility.Obstetrician/gynecologists have known for quite a while that as women age, their risk of miscarriage increases, as does the risk of having children with Down syndrome or other genetic defects. Advanced paternal age has also been implicated in a range of reproductive and genetic abnormalities, from reduced fertility to some diseases of complex etiology such as schizophrenia. This research team has previously reported that as men age, their sperm counts decline and their sperm become less active. However, the mechanisms for the effect of older paternal age on genetic defects are not well understood.In this study, the researchers analyzed the sperm of 97 men after an average of 5.1 days of sexual abstinence. The men were nonsmokers aged 22–80 who were in generally good health. The researchers used flow cytometry and statistical analysis to observe associations between a subject’s age, his semen quality, and genomic abnormalities in his sperm such as DNA fragmentation, aneuploidy, diploidy, and mutations related to achondroplasia and Apert syndrome.Increased age of the men was not associated with the same genetic defects seen in older women; for instance, there was no increased risk of fathering a child with Down syndrome. But some older fathers did have an increased risk of having children with achondroplasia, and according to the published results, ”a small fraction of men are at increased risk for transmitting multiple genetic and chromosomal defects.” The authors caution that their findings are preliminary and are based on a small number of tests in a small population of men. However, they believe their findings suggest that as men age, they may have more difficulty fathering children.FiguresReferencesRelatedDetails Vol. 114, No. 9 September 2006Metrics About Article Metrics Publication History Originally published1 September 2006Published in print1 September 2006 Financial disclosuresPDF download License information EHP is an open-access journal published with support from the National Institute of Environmental Health Sciences, National Institutes of Health. All content is public domain unless otherwise noted. Note to readers with disabilities EHP strives to ensure that all journal content is accessible to all readers. However, some figures and Supplemental Material published in EHP articles may not conform to 508 standards due to the complexity of the information being presented. If you need assistance accessing journal content, please contact [email protected]. Our staff will work with you to assess and meet your accessibility needs within 3 working days.

The flow cytometric analysis of premalignant and malignant lesions in head and neck squamous cell carcinoma

We attempted to identify the molecular mechanisms involved in Head and Neck Squamous Cell Carcinoma (HNSCC) pathogenesis by measuring the nuclear DNA content (ploidy) in premalignant (potentially malignant) and malignant patients as compared to normal controls, and to determine whether DNA ploidy could be used to predict the clinical outcome. From March 2001 to December 2003, the analysis was carried out in a set of 41 patients with premalignant lesions and 79 suffering from squamous cell carcinoma of laryngeal, oesophageal, nasopharyngeal, nasal and oral lesions and 50 controls. Representative samples were taken by punch biopsy and processed using standard formol-paraffin technique for histopathological examination. Fifty micrometer thick sections of paraffin-embedded tissues were analyzed to detect the DNA content by image cytometry. Of the potentially malignant patients, 46% had diploid lesions, 37% had tetraploid lesions and 17% had aneuploid lesions. While of the patients with cancer, 90% had aneuploid lesions, 10% had diploid lesions and none had tetraploid lesions. DNA diploidy tended to occur earlier in the progression from premalignant to malignant lesions and this helps us early detection of HNSCC by DNA from lesions in high risk groups and examination of its ploidy. Knowledge of tumor cell ploidy by DNA image cytometry may facilitate the evaluation of malignant and premalignant lesions in HNSCC. The present findings are promising to supplement clinical and histopathological parameters in evaluating prognosis and to demonstrate methods that are readily applicable for routine diagnostic work.

Prognostic Biomarkers in Renal Cell Carcinoma: Relevance of DNA Ploidy in Predicting Disease-Related Survival

To investigate the prognostic value of DNA ploidy, Ki-67 index and p53 expression in relation to disease-related survival in a consecutive series of patients with renal cell carcinoma (RCC). The study group consisted of 64 RCC patients treated by radical nephrectomy. Histological type, pathological staging and nuclear anaplasia were assessed according to the WHO classification, TNM system and Fuhrman grading criteria, respectively. Ploidy was determined by DNA flow cytometry using two sampling methods (frozen vs paraffin-embedded tissue). Ki-67 and p53 were evaluated by immunohistochemistry techniques using two cutoff points (10% vs mean value) for staining interpretation. Kaplan-Meier and Cox regression analyses were used for prognostic evaluation. Thirty-one tumors (48.4%) showed DNA diploidy and 33 (51.6%) were DNA aneuploid. Concordance between both ploidy measurement methods was found in 85.5% of cases (p=0.0455). The mean values for Ki-67 and p53 immunostaining were 3.65% (0-23.5%) and 5.90% (0-55.9%), respectively. DNA ploidy significantly correlated with staging, tumor size (pT), nuclear grading, and Ki-67 (mean value cutoff). Ki-67 (10% cutoff) correlated with staging and pT, while p53 (mean value cutoff) was associated with Ki-67 (mean value cutoff). There were significant differences between survival curves for pathological stage, pT, nuclear grade, ploidy, Ki-67 (both cutoffs), and p53 (10% cutoff). By univariate regression analysis, stage III and stage IV, pT3, aneuploidy, high Ki-67 (both cutoffs), and p53 overexpression (10% cutoff) showed significant correlations with worse disease-related survival. In addition, DNA aneuploidy significantly correlated with poor prognosis within stages I/II (p=0.0355) and stages III/IV (p=0.0138) of the disease. The results indicate that DNA ploidy has relevant prognostic value in RCC, adding useful information to the classic histopathological indicators of clinical outcome.

Nodular Clear Cell Hidradenoma: A Case Report with Immunohistochemistry, Cytogenetics and Flow Cytometry

Background: Nodular Hidradenoma (NH) is an uncommon neoplasm with controversial cell origin (myoepithelial, eccrine and more recently apocrine), and few cytogenetic and DNA flow Cytometry data. Material and Methods: We report a case of NH on the left ankle of a 77 year‐old man. The tumor was located subcutaneously and measured 2.5 × 2.0 cm, with no throbbing, tenderness, edema, or groin lymphadenopathy. A wide local excision was performed. Immunohistochemistry using monoclonal antibodies to Smooth Muscle Actin (SMA), S‐100 protein, p63 and Calponin were examined. A fresh central portion of the tumor was examined for karyotyping and DNA flow cytometry. Six months later the patient was healthy without local recurrence. Results: The tumor was positive for SMA, Calponin and p63 with focal staining for S‐100. The karyotype showed four aberrant, unrelated clones, t(1;8)(p22;p23), del(11)(q14q23), t(1;10)(p13;q22) and del (6)(q23q26). DNA flow cytometry and cell cycle analysis showed that the tumor is 100% diploid with 6.55% S‐phase. Conclusions: Expression of SMA, p63, Calponin, and S‐100, which are myoepithelial markers, support a myoepithelial origin of this tumor. The multiclonal pattern observed in this neoplasm seems to reflect cytogenetic divergence during neoplastic progression. DNA diploidy with a low S‐phase predicts a benign clinical course.

Multicolor FISH and cytometric analyses allow classification of urothelial carcinomas into two subtypes, low- and high-grade tumors.

The biological behaviors of urothelial carcinomas are linked roughly to histological grade, but there are many cases whose precise classification is difficult. In addition, it remains unclear whether there are differences in the types of genetic changes between low- and high-grade urothelial carcinomas. Fifty-seven biopsy specimens of urothelial carcinomas and 9 control urothelial specimens were examined by four-color FISH with centromere-specific probes for chromosomes 3, 7, and 17, and a locus-specific probe for 9p21 that covers p16INK4a and p15INK4b. Nuclear DNA ploidy was determined with laser scanning cytometry (LSC). FISH and LSC data allowed us to classify urothelial cancers into two groups. Tumors in one group showed minimal intercellular variation in centromere copy numbers and DNA diploidy, and these tumors were histologically comparable to low-grade (grades I and II) tumors. In contrast, tumors in the other group showed a large intercellular variation in centromere copy numbers and DNA aneuploidy, suggesting chromosomal instability. These tumors were all high-grade (grades II and III) tumors. Numerical abnormalities of 9p21 signals were detected in all cancers; this variation in 9p21 signals was also associated with histological grade. The present findings suggest that the pathogenetic pathways are different between low- and high-grade carcinomas. High-grade tumors are characterized by chromosomal instability, whereas low-grade carcinomas are not.

Stratification of the biologic aggressiveness of non-small cell lung cancer using DNA flow cytometry and immunohistochemistry for the retinoblastoma protein

DNA ploidy pattern and S-phase fraction (SPF) measured by flow cytometry and expression of the retinoblastoma gene product (pRB) estimated by immunohistochemistry were correlated with outcome in 114 patients who received a curative operation for primary non-small cell lung cancer (NSCLC). One hundred ten tumors yielded an adequate DNA histogram, and all tumors exhibited an assessable immunohistochemical stain. DNA diploidy was detected in 31 tumors and DNA aneuploidy in 79 tumors. The mean SPF was 14.1±6.4%. Eighty tumors were positively stained, and 34 tumors were negative for pRB. Multivariate analysis clarified that both TNM staging and DNA ploidy were prognostic factors after surgery. In 39 recurrent cases, the SPF value was inversely correlated with disease-free interval. With only supportive care after recurrence, high SPF tumors and pRB-negative tumors progressed rapidly, whereas active treatment yielded an equivalent effect on recurrent tumors regardless of the SPF or pRB expression. DNA ploidy is related to the risk of recurrence, while SPF is related to tumor growth rate and the impact of active treatment on recurrence. The utility of pRB expression was limited. The combination of DNA ploidy and SPF allows practical stratification of the biologic aggressiveness of NSCLC.

Stratification of the biologic aggressiveness of non-small cell lung cancer using DNA flow cytometry and immunohistochemistry for the retinoblastoma protein

<h2>Abstract</h2> DNA ploidy pattern and S-phase fraction (SPF) measured by flow cytometry and expression of the retinoblastoma gene product (pRB) estimated by immunohistochemistry were correlated with outcome in 114 patients who received a curative operation for primary non-small cell lung cancer (NSCLC). One hundred ten tumors yielded an adequate DNA histogram, and all tumors exhibited an assessable immunohistochemical stain. DNA diploidy was detected in 31 tumors and DNA aneuploidy in 79 tumors. The mean SPF was 14.1±6.4%. Eighty tumors were positively stained, and 34 tumors were negative for pRB. Multivariate analysis clarified that both TNM staging and DNA ploidy were prognostic factors after surgery. In 39 recurrent cases, the SPF value was inversely correlated with disease-free interval. With only supportive care after recurrence, high SPF tumors and pRB-negative tumors progressed rapidly, whereas active treatment yielded an equivalent effect on recurrent tumors regardless of the SPF or pRB expression. DNA ploidy is related to the risk of recurrence, while SPF is related to tumor growth rate and the impact of active treatment on recurrence. The utility of pRB expression was limited. The combination of DNA ploidy and SPF allows practical stratification of the biologic aggressiveness of NSCLC.

Improved grading of breast adenocarcinomas based on genomic instability.

Numerous investigations have shown that in primary breast adenocarcinomas DNA aneuploidy in contrast to DNA diploidy indicates high malignancy potential. On the basis of the study of 104 breast carcinomas, we describe a subtype of aneuploidy, which demonstrates a low degree of malignancy. In image cytometric DNA histograms, this subtype possessed a low percentage (< or = 8.8%) of nonmodal DNA values as measured by the stemline scatter index (SSI), which is defined as sum of the percentage of cells in the S-phase region, the G(2) exceeding rate and the coefficient of variation of the tumor stemline. The cut point of SSI = 8.8% (P = 0.03) enabled us to also subdivide diploid and tetraploid tumors into clinically low and high malignant variants. One possible reason for aneuploidy is impaired distribution of chromosomes at mitosis caused by numerical or structural centrosome aberrations. Cyclins A and E seem to be involved in centrosome duplication. Real-time quantitative PCR measurements of cyclin A and E transcript levels and immunohistochemical determination of cyclin A protein expression showed statistically significantly increased values in the tumors with a high SSI (>8.8%), compared with those with a low SSI. A pilot study demonstrated centrosomal aberrations in an average of 9.6% of the measured cells in four aneuploid carcinomas with high SSI values and in an average of 2.5% of the cells in three aneuploid and three diploid tumors with low SSI. Our data indicate that the SSI, most likely reflecting the degree of genomic instability, allows additional classifying of the known aneuploid, diploid, and tetraploid categories of primary breast adenocarcinomas into low and high malignant subtypes.

Cyclo-oxygenase-2 (COX-2) mRNA expression correlates with progesterone receptor positivity in human breast cancer.

We previously presented evidence showing that cyclo-oxygenase 2 (COX-2) plays an important role in mammary carcinogenesis and angiogenesis in human breast cancer. The present study aims to compare COX-2 mRNA expression with hormone receptor status, S-phase fraction, telomerase activity, and DNA ploidy in human breast cancer. Total cellular RNA was extracted from frozen breast tissue samples according to standard methodology. The mRNA copy numbers for COX-2 were determined in 18 infiltrating carcinomas using quantitative RT-PCR and TaqMan methodology. The oestrogen receptor (ER) and progesterone receptor (PgR) status was determined using the ligand-binding technique (ER+ = > 3 fmol/mg, PgR+ = > 5 fmol/mg). We also determined DNA ploidy status (diploid or aneuploid), S-phase fraction (< 6% = low, 6-10% = intermediate, > 10% = high), and telomerase activity (total protein generated by TRAP assay). The median COX-2 mRNA copy number per micro g of RNA was 126 713 (range = 15 717-2 022 050). COX-2 expression was significantly associated with PgR positivity (p = 0.013). The association between COX-2 and DNA diploidy failed to reach a statistical significance (p = 0.085). No significant association was detected between COX-2 and S-phase fraction, ER status, or telomerase activity. COX-2 mRNA expression is associated with PgR positivity in human breast cancer. This observation is consistent with the hypothesis that COX-2 upregulates aromatase activity.

Cyclo-oxygenase-2 (COX-2) mRNA expression correlates with progesterone receptor positivity in human breast cancer

SUMMARYBackground: We previously presented evidence showing that cyclo-oxygenase 2 (COX-2) plays an important role in mammary carcinogenesis and angiogenesis in human breast cancer. The present study aims to compare COX-2 mRNA expression with hormone receptor status, S-phase fraction, telomerase activity, and DNA ploidy in human breast cancer.Methods: Total cellular RNA was extracted from frozen breast tissue samples according to standard methodology. The mRNA copy numbers for COX-2 were determined in 18 infiltrating carcinomas using quantitative RT-PCR and TaqMan methodology. The oestrogen receptor (ER) and progesterone receptor (PgR) status was determined using the ligand-binding technique (ER+ = >3 fmol/mg, PgR+ = >5fmol/mg). We also determined DNA ploidy status (diploid or aneuploid), S-phase fraction (<6% = low, 6-10% = intermediate, > 10% = high), and telomerase activity (total protein generated by TRAP assay).Results: The median COX-2 mRNA copy number per ug of RNA was 126 713 (range = 15717-2022050). COX-2 expression was significantly associated with PgR positivity (p = 0.013). The association between COX-2 and DNA diploidy failed to reach a statistical significance (p = 0.085). No significant association was detected between COX-2 and S-phase fraction, ER status, or telomerase activity.Conclusions: COX-2 mRNA expression is associated with PgR positivity in human breast cancer. This observation is consistent with the hypothesis that COX-2 upregulates aromatase activity.