DNA Histogram Analysis Research Articles

DNA image cytometric analysis of bronchial washings as an adjunct for the detection of lung cancer in a clinical setting.

BackgroundDNA aneuploidy has a potential to become an adjunct to conventional cytology for diagnosis of lung cancer, but its value in bronchial washings has not been well evaluated.MethodsWe conducted a retrospective study on patients who underwent bronchoscopy and the bronchial washings were submitted for both cytology and DNA image cytometry (DNA‐ICM) examination. The sensitivity and specificity of two methods and both in combination were compared. Analysis of clinical subgroups and DNA histogram were also performed.ResultsThe study included 626 patients (326 patients with lung cancer and 300 patients with benign lung diseases). The sensitivity of cytology, DNA‐ICM, and combination test for lung cancer were 53.3%, 62.3%, and 75.8%, respectively, and the sensitivity of DNA‐ICM and combination test were superior to that of cytology (p < 0.05). A modest reduction of specificity was found in DNA‐ICM compared with cytology (91.3% vs. 98.3%, p < 0.05). Subgroup analysis showed there was no significant difference in sensitivity of DNA‐ICM between the visible tumor group and the invisible tumor group (66.5% vs. 56.9%, χ2 = 3.114, p = 0.078). Among 101 patients with invisible endobronchial tumor, the positive rates for DNA‐ICM of washing, cytology of washing, brushing and biopsy were 62.4%, 41.6%, 40.6%, and 45.5%, respectively. DNA‐ICM in combination with the basic bronchoscopy techniques could increase the sensitivity from 67.3% to 87.1% (p = 0.000). The DNA histogram analysis showed 25.3% washing samples of lung cancer were diploid pattern, 49.4% were scattered aneuploid cells pattern, and 25.3% were aneuploid peaks pattern. Small cell lung cancer had the highest proportion of aneuploid peaks pattern (p < 0.05).ConclusionsDNA‐ICM could be used as an adjunct for the detection of lung cancer. The combination of DNA‐ICM and basic bronchoscopy techniques could significantly increase the sensitivity, especially for the patients suspected of peripheral lung cancer, and contribute to select subjects for advanced bronchoscopy.

Cordycepin, isolated from medicinal fungus Cordyceps sinensis, enhances radiosensitivity of oral cancer associated with modulation of DNA damage repair

Concurrent chemotherapy and radiotherapy (RT) is important for controlling oral squamous cell carcinoma (OSCC), which is often accompanied by significant acute and late toxicities. We investigated whether cordycepin, a small molecule extracted from Cordyceps sinensis, could enhance the radiosensitivity of oral cancer cells. Using colony formation assay, we demonstrated that cordycepin induces radiosensitizing effects on two OSCC cells. DNA histogram analysis showed that cordycepin combined with RT prolonged the RT-induced G2/M phase arrest. It protracted the duration of DNA double strand breaks, which was detected by immunofluorescent staining of phosphorylated histone H2AX (γ-H2AX). The underlying molecular mechanism might involve the downregulation of protein expression related to DNA damage repair, including phosphorylated ataxia-telangiectasia mutated (p-ATM) and phosphorylated checkpoint kinase 2. Reciprocal upregulation of phosphorylated checkpoint kinase 1 (Chk1) expression was noted, and the radiosensitizing effect of cordycepin could be further augmented by Chk1 mRNA knockdown, indicating a compensatory DNA repair machinery involving phosphorylation of Chk1. In vivo, the combination of cordycepin and RT exhibited greater growth inhibition on xenografts and stronger apoptosis induction than RT alone, without exacerbating major toxicities. In conclusion, cordycepin increased the radiosensitivity of OSCC cells, which is associated with the modulation of RT-induced DNA damage repair machinery.

Diamine and PEGylated-diamine conjugates of triterpenic acids as potential anticancer agents

A set of 18 amide derivatives of oleanolic or maslinic acid has been semi-synthesised. Twelve were diamine conjugates at C-28 of these triterpenic acids and the other six were PEGylated-diamine derivatives. The cytotoxic effects of these 18 triterpenic derivatives in three cancer-cell lines (B16-F10, HT29, and Hep G2) have been assayed, and have been compared to three non-tumour cell lines of the same or a similar tissue (HPF, IEC-18, and WRL68). The cell viability percentages for the non-tumour HPF line for almost all diamine conjugates of the tested triterpenic acids ranged from 81% to 94%. The best cytotoxic results were achieved with the diamine conjugates of oleanolic or maslinic acid with the shortest and the longest diamine chain (IC50 values from 0.76 μM to 1.76 μM), on the B16-F10 cell line, being between 140- and 20-fold more effective than their corresponding precursors. Four diamine conjugates of these triterpenic acids showed apoptotic effects on treated cells of the B16-F10 line, with total apoptosis rates, relative to control, of between 73% and 90%. The DNA-histogram analysis revealed that all compounds tested produced cell-cycle arrest in B16-F10 cells, increasing the number of these cells in the S phase. All the compounds analysed, except one, did not cause changes in mitochondrial-membrane potential during apoptosis of the B16-F10 cancer cells, suggesting an activation of the extrinsic apoptotic pathway for these compounds.

Molecular prognostic factors in early-stage cervical adenocarcinoma.

Within the past years the proportion of cervical adenocarcinomas has increased, however, there is a shortage of data regarding immunohistochemical and molecular features and their prognostic relevance in early-stage cervical adenocarcinoma (esCAC). Aim of the present study was to evaluate molecular prognostic factors in esCAC patients treated with primary surgery. Analyses of surgical specimens in 59 patients with esCAC were performed on fixed paraffin-embedded sections of tumour tissue. Tumour tissue sections were routinely stained with hematoxylin and eosin followed by microscopic examination. Immunohistochemical analyses (IHC) were performed on paraffin-embedded section. Flow cytometry (FCM) analysis of paraffin-embedded tumor tissue was performed using flow cytometer FACSCalibur equipped with argon laser. DNA histogram analysis was performed with ModFit application. Treatment effectiveness was evaluated using overall 5-year survival. Survival probability was estimated using the Kaplan-Meier method. Overall survival rate estimated using Kaplan-Meier method was 74.6%. Among the IHC and FCM features univariate analysis showed statistical significance of nm23-H1 gene expression and total S-phase fraction ≤ 11.9% (S-TOT). In multi- variate analysis LVSI and parametrial involvement had significant, negative impact on survival (HR = 8.04, p < 0.003 and HR = 4.03, p < 0.017, respectively). However, none of the tested IHC and FCM features had any influence on overall 5-year survival.

Cyclin D1 promoter -56 and -54 bp CpG un-methylation predicts invasive progression in arsenic-induced Bowen’s disease

BackgroundPatients with arsenic-induced Bowen’s disease (As-BD) are at risk of developing invasive cancers in the skin, lung, and urinary bladder. However, a longitudinal follow-up study on the association between As-BD and invasive cancers is still lacking. ObjectivesThis study aims to investigate the underlying molecular mechanisms of this malignant progression in the skin and internal organs. MethodsThis is a biopsy-based follow-up study. We tested the DNA histograms, Cyclin D1 (CCND1) protein expression and CCND1 promoter DNA methylation in 40 pathologically confirmed specimens from As-BD patients to correlate with individual’s invasive cancer occurrence in the 5-year follow-up. ResultsFlow cytometric DNA histogram analysis of skin specimens showed aneuploid (n=15), G2/M arrest (n=22), and normal (n=3) DNA histograms. No patients with normal DNA histograms developed invasive cancers, whereas 13 developed invasive cancers in the aneuploid group and 2 developed invasive cancers in the G2/M arrest group. The aneuploid group showed a high risk of invasive cancer development. In all assessed aneuploid specimens, the CCND1 promoter hypomethylation was observed. Statistically, percentage of un-methylation more than 55.85% among 17 detected CpG sites showed extremely high predictive power in the occurrence of invasive arsenical cancers. Furthermore, the un-methylation at -56 and -54bp CpG sites was statistically significantly associated with invasive arsenical cancer development (p=1.29×10−5). ConclusionsAs-BD lesions showing an aneuploid DNA histogram had a high risk of invasive cancer development. Un-methyaltion at -56 and -54bp CpG in the CCND1 promoter serves as a predictor for invasive progression in As-BD patients.

Quick cytogenetic screening of breeding bulls using flow cytometric sperm DNA histogram analysis.

The aim of the present study was to test the FXCycle PI/RNase kit for routine DNA analyses in order to detect breeding bulls and/or insemination doses carrying cytogenetic aberrations. In a series of experiments we first established basic DNA histogram parameters of cytogenetically healthy breeding bulls by measuring the intraspecific genome size variation of three animals, then we compared the histogram profiles of bulls carrying cytogenetic defects to the baseline values. With the exception of one case the test was able to identify bulls with cytogenetic defects. Therefore, we conclude that the assay could be incorporated into the laboratory routine where flow cytometry is applied for semen quality control.

SRJ23, a new semisynthetic andrographolide derivative: in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis in prostate cancer cells.

3,19-(3-Chloro-4-fluorobenzylidene)andrographolide (SRJ23), a new semisynthetic derivative of andrographolide (AGP), exhibited selectivity against prostate cancer cells in the US National Cancer Institute (NCI) in vitro anti-cancer screen. Herein, we report the in vitro growth inhibition and mechanisms of cell cycle arrest and apoptosis induced by SRJ23. 3-(4,5-Dimethythiazol-2-yl)-2,5-diphenyl tetrazolium bromide (MTT) assay was used in assessing in vitro growth inhibition of compounds against prostate cancer (PC-3, DU-145 and LNCaP) and mouse macrophage (RAW 264.7) cell lines. Flow cytometry was utilised to analyse cell cycle distribution, whereas fluorescence microscopy was performed to determine morphological cell death. DNA fragmentation and annexin V-fluorescein isothiocyanate (FITC)/propidium iodide (PI) flow cytometry were done to confirm apoptosis induced by SRJ23. Quantitation of cell cycle and apoptotic regulatory proteins were determined by immunoblotting. AGP and SRJ23 selectively inhibited the growth of prostate cancer cells compared with RAW 264.7 cells at low micromolar concentrations; however, SRJ23 was more potent. Mechanistically, SRJ23-treated PC-3 cells displayed down-regulation of cyclin-dependent kinase (CDK) 1 without affecting levels of CDK4 and cyclin D1. However, SRJ23 induced down-regulation of CDK4 and cyclin D1 but without affecting CDK1 in DU145 and LNCaP cell lines. DNA histogram analysis revealed that the SRJ23 induced G2/M in PC-3 cells but G1 arrest in DU-145 and LNCaP cells. Morphologically, both compounds induced predominantly apoptosis, which was further confirmed by DNA fragmentation and annexin V-FITC staining. The DNA fragmentation was inhibited in the presence of caspase 8 inhibitor (Z-IETD-FMK). Apoptosis was associated with an increase in caspase 8 expression and activation. This thought to have induced cleavage of Bid into t-Bid. Additionally, increased expression and activation of caspase 9 and Bax proteins were apparent, with a concomitant down-regulation of Bcl-2 protein. Similar apoptosis cascade of events was observed in SRJ23-treated DU145 and LNCaP cell lines. SRJ23 inhibited the growth of prostate cancer cells by inducing G2/M and G1 arrest via down-regulation of CDK1, and CDK4 and cyclin, respectively, and initiated caspase-8-mediated mitochondrial apoptosis. Taken together, these data support the potential of this compound as a new anti-prostate cancer agent.

Bacteriostatic versus bactericidal activity of ciprofloxacin in Escherichia coli assessed by flow cytometry using a novel far-red dye

As common microbiological methods for the assessment of bacteriostatic or bactericidal activities are very time-consuming, in this work we describe that the use of a novel far-red fluorescent stain, Vybrant DyeCycle Ruby (DCR) for the flow cytometric analysis of fluoroquinolone (ciprofloxacin) bacteriostatic and bactericidal activities in Escherichia coli proved to be specific for bacterial DNA and, after ciprofloxacin exposure, DNA distribution analysis was achieved using a 7.5 μM DCR concentration to stain 5 × 10⁵ ethanol-fixed bacterial cells. The analysis of the bacterial DNA histograms obtained from the ciprofloxacin concentrations tested, enabled the distinction between ciprofloxacin bacteriostatic and bactericidal activities.

Transactivation of RON receptor tyrosine kinase by interaction with PDGF receptor β during steady-state growth of human mesangial cells

Although it is well known that platelet-derived growth factor (PDGF) causes mesangial cell proliferation (presumably contributing to progression of glomerular disease), targeted inhibition of the PDGF receptor system has shown only limited efficacy against glomerular diseases. To examine whether this discrepancy is due to the involvement of other pathways, we used phosphorylated receptor tyrosine kinase arrays and found that RON (recepteur d'origine nantais) was phosphorylated while the PDGF receptor was dephosphorylated (thus inactive) in human mesangial cells (HMCs) at the time of cell cycle entry. Further, RON remained active during steady-state growth. Activation of RON was independent of its canonical ligand, macrophage-stimulating protein, but was mediated by transactivation from the PDGF-engaged PDGF receptor. Following stimulation with PDGF we found that the two receptors physically interacted. Knockdown of RON by siRNA increased the number of apoptotic cells without affecting the rate of DNA synthesis, suggesting that RON has anti-apoptotic functions. Immunohistochemical analysis found phosphorylated RON in glomerular lesions of patients with IgA nephropathy but not those with minimal change nephrotic syndrome, a disease not associated with mesangial proliferation. These results suggest that RON is involved in mesangial cell proliferation under both physiological and pathological conditions, and may be a relevant target for therapeutic intervention.

Photodynamic Therapy Decreases DNA Content Abnormalities in Residual Non-Dysplastic Barrettʼs Esophagus

Purpose: Photodynamic therapy (PDT) has been used for the treatment of Barrett's esophagus (BE) with high grade dysplasia (HGD) or early adenocarcinoma (AC). After PDT, some patients still have residual surface BE (SBE) and/or buried BE (BBE) underlying neosquamous epithelium. The neoplastic potential of residual SBE and BBE post-PDT is unknown. Methods: 52 BE patients with HGD or early AC were treated with PDT. Pre-PDT and post-PDT biopsies were performed. 22 matched pre- and post-PDT non-dysplastic BE samples were retrieved from 11 patients for high fidelity DNA histogram analysis. DNA content changes were compared between pre-PDT SBE, post-PDT SBE and BBE samples. DNA index > 1.1 was considered aneuploid. Results: Of the 11 pre-PDT SBE samples, 3 were diploid and 8 were aneuploid (Table 1). In the 3 patients with pre-PDT SBE diploidy, the post-PDT residual SBE and BBE samples were also diploid. Interestingly, in the 8 patients with pre-PDT SBE aneuploidy, all the post-PDT BBE and 5 post-PDT SBE samples were diploid; 2 post-PDT SBE samples remained aneuploid, and 1 was not analyzed because of insufficient tissue. Conclusion: Compared to pre-PDT BE, post-PDT BE shows significantly less frequent DNA content abnormalities, suggesting that PDT may have a preferential destructive effect on aneuploid cells. This indicates that the post-PDT residual BE, both surface and buried, may have a lower neoplastic potential than pre-PDT BE.Table: DNA content changes in non-dysplastic Barrett's esophagus before and after photodynamic therapy

DNA Image Cytometry Is a Useful Adjunct Tool in the Prediction of Disease Outcome in Patients with Stage II and Stage III Colorectal Cancer

Background: We assessed the prognostic value of the nuclear DNA content measured in the primary tumours of 123 patients with stage II or stage III colorectal cancer (CRC). Methods: Isolated nuclei from paraffin sections were stained with the Feulgen reaction, and DNA was measured using a computer-assisted image analysis cytometry system. We applied 4 different approaches in the analysis of DNA histograms: the ABCDE approach, histogram range, peak evaluation and DNA cut-off values. Results: Using the histogram range, the narrow range was rare (3.7%) in patients who died of disease (n = 28) as compared with 16.4% among those alive (n = 74; p = 0.017). Modal peak evaluation was a significant predictor of disease-free survival (DFS; Kaplan-Meier log-rank p = 0.0235). In the range evaluation, the 1st set (low-start gates) was a significant predictor of DFS (log-rank p = 0.0121), where disease recurrence was closely associated with the widest range (1.8–>10c; c = haploid DNA content) gates. Recurrence-free survival was 3 times better in narrow-gate histograms than wide-range histograms (p < 0.03). The 1st set also proved to be a significant predictor of disease-specific survival (DSS; log-rank p = 0.0045), which was markedly better (77.8–90.0%) among the patients with the narrow-gate histograms. Grading of the histogram range into 2 categories (with 6.0c as cut-off), was a powerful predictor of both DSS (log-rank p = 0.0092) and 5-year DFS (p = 0.0106) in the whole series, and separately in stage III (but not stage II) disease, with p = 0.0131 and p = 0.0201, respectively. Conclusion: The DNA image cytometry with careful analysis of the histograms may provide valuable prognostic information in CRC, with potential clinical implications in patient management, particularly in predicting the patients at high risk for recurrence who should be considered as candidates for adjuvant therapy.

DNA Image cytometry as a prognostic tool in stage II and stage III colorectal cancer

13565 Background: We assessed the prognostic value of nuclear DNA content measured in the primary tumors of 123 patients with stage II or stage III colorectal cancer (CRC). Methods: Isolated nuclei from paraffin sections were stained with Feulgen and DNA was measured using a computer-assisted image analysis cytometry system (Ahrens ACAS). We applied 4 different approaches in analysis of DNA histograms: ABCDE approach, histogram range, peak evaluation, and DNA cut-off values. Results: Using the histogram range, narrow range was rare (3.7%) in patients who died of disease as compared with 16.4% among those alive (p=0.017). Modal peak evaluation was a significant predictor of disease free survival (DFS) (Kaplan-Meier log-rank p=0.0235). In the range evaluation, the first set (low-start gates) was a significant predictor of DFS (log-rank p=0.0121), where disease recurrence was closely associated the widest range (1.8c-&gt;10c) gates. Recurrence-free survival was markedly better among patients with narrow gate histograms than wide range histograms than among patients with wide range histograms (p&lt;0.03). The first set also proved to be significant predictor of disease specific survival (DSS) (log-rank p=0.0045), being markedly better (78–90.0%) among the patients with the narrow-gate histograms. Grading of the histogram range into two categories (with 6.0c as cut-off for low and wide range), was a powerful predictor of both DSS (log-rank p= 0.0092) and 5-year DFS (p=0.0106) in the whole series, and separately in Stage III (but not Stage II) disease; p=0.0131 and p=0.0201, respectively. Conclusions: The DNA image cytometry with careful analysis of the histograms may provide valuable prognostic information in CRC, with potential clinical implications in patient management, particularly in predicting the patients at high risk for recurrence who should be considered as candidates for adjuvant therapy. No significant financial relationships to disclose.

Decreased Phosphoactive ERKs and JNKs in Malachite-Green-Transformed Syrian Hamster Embryo Fibroblasts Are Associated with Increased Phosphoactive p38 Kinase: Possible Therapeutic Importance

Background: Malachite green (MG), consisting of green crystals with a metallic lustre, is highly soluble in water, cytotoxic to various mammalian cells and also acts as a liver tumor promoter. In view of its industrial importance and possible exposure of human beings, MG poses a potential environmental health hazard. We have earlier reported the malignant transformation of Syrian hamster embryo (SHE) cells by MG. Methods: Cell transformation assays were carried out as described in the literature. Western blotting and flow cytometry were carried out by standard methods. Results: In this study, we have studied the role of all three isoforms of mitogen-activated protein (MAP) kinases, i.e. extracellular regulated kinases (ERKs), Jun N-terminal kinases (JNKs) and p38 kinase in the MG-transformed SHE fibroblasts compared to controls. Our results showed that transformed cells were associated with decreased expression of ERKs and JNKs as evidenced by Western blotting studies. However, the p38 MAP kinase was found to be upregulated. Flow cytometric DNA histogram analysis indicated an increase in the expression of S phase cells in the transformed cell line as compared to their control counterparts. Conclusions: The present studies indicate that decreased phosphoactive ERKs and JNKs and increased phosphoactive p38 kinase are associated with increased S phase cells during transformation of SHE cells by MG.

Levels of apoptosis in human granulosa cells seem to be comparable after therapy with a gonadotropin-releasing hormone agonist or antagonist

To compare levels of apoptosis in granulosa cells from women treated with the gonadotropin-releasing hormone (GnRH) agonist triptorelin or the GnRH antagonist cetrorelix. Randomized, prospective study. University hospital. Thirty-two women undergoing assisted reproduction techniques after ovulation induction with recombinant follicle-stimulating hormone (FSH) plus GnRH agonist or antagonist. Granulosa cells were isolated from follicular aspirates after oocyte removal. Apoptosis was assessed with Annexin V binding assay, terminal deoxynucleotidyl transferase (TdT)-mediated nick-end labeling (TUNEL) assay, flow cytometric analysis of DNA, and ultrastructural analysis of cell morphology in transmission electron microscopy. Serum and follicular hormonal levels were also determined. Annexin V binding and TUNEL assays revealed comparable percentages of apoptosis in the two groups under investigation. Analysis of DNA histograms revealed a similar cell cycle distribution in the two groups. Ultrastructural analysis only occasionally displayed patterns of chromatin margination in apoptotic cells. The mean concentrations of all the follicular fluid steroid hormones evaluated (E2, T, and P) were significantly lower in the GnRH antagonist-treated group. Therapy with a GnRH agonist or antagonist is associated with comparable levels of apoptosis in granulosa cells.

DNA histogram analysis for node-negative breast cancer.

The combination of DNA ploidy and S-phase is one of the strongest general prognostic indicators for node-negative breast cancer (1). The costs associated with this prognostic power are relatively minimal; laboratory total processing time is usually less than 10 min and reagents are relatively inexpensive. The test can be standardized world-wide as long as strict guidelines are followed, and the test’s result can be presented in a way that is easily understood by oncologists. DNA ploidy and S-phase prognostic strength can be further augmented by other wellknown prognostic indicators, such as menopausal status and primary tumor size. Figures 1 and 2 provide evidence for the above statements (1). Figure 1A shows highly significant relapse-free survival (RFS) stratifications for over 935 node-negative patients in the Baylor study. The stratifications are based on a prognostic model composed of an optimal combination of adjusted DNA ploidy and S-phase. When the same prognostic model and stratification boundaries are applied to other independent studies (see Fig. 1B and C), similar highly-significant patient stratifications are observed. Figure 2 demonstrates how the prognostic model can be further augmented by primary size and menopausal status. These patient stratifications are potentially important for managing this prevalent and deadly disease. In 1999, the incidence of breast cancer in the United States was over 170,000; greater than leukemia, lymphoma, and AIDS combined. In 2003, it is predicted to be over 200,000. Most of these cases are discovered before the tumor has infiltrated into the patient’s nodes (node-negative); therefore, this diagnostic test can potentially impact the strategy for optimal treatment for this large patient group. The DNA histogram test can be divided into four processing steps: sample preparation, DNA histogram acquisition, cell-cycle analysis, and final processing. In order for cytometrists to perform this test to its full capability, the following guidelines for each of these steps are recommended.

Effects of resolution reduction on data analysis.

There is often a need in flow cytometry to display and analyze histograms at resolutions lower than those native to the data. It is common, for example, to analyze DNA histograms at 256-channel resolution, even though the data were acquired at 1,024 channels or more. The most common method for reducing resolution, referred to as the consecutive summation (CS) method, can introduce distortions into the shape of histograms. Peaks that were symmetric in the original data can become skewed in the reduced-resolution histogram. Data analysis can be negatively affected by the distortions produced by reducing the histogram resolution. An alternative technique for reducing histogram resolution, the unbiased summation (US) method, minimizes shape distortion. This paper describes the US method and examines the benefits it provides in the analysis of DNA histograms. Reduced chi-square (RCS) was used to measure the response to three experimental variables in the least-squares analysis of simulated DNA histograms. For each variable (the percentage of coefficient of variation [%CV], number of events, and mean position of the G1 distribution), a test data set of 1,000 histograms was generated at 1,024-channel resolution. Histogram resolutions were reduced with each method and then analyzed with ModFit LT cell-cycle analysis software (Verity Software House, Topsham, ME). S-phase error and processor computation time of each method also were evaluated. A Monte Carlo experiment was performed to compare CS and US methods to theoretically correct reductions. CS method analysis results were negatively affected by changes in %CV, number of events, and G1 peak position. The US method produced consistently lower RCS values (more accurate results) within the tested ranges. The US method eliminated bias in S-phase error and had negligible impact on analysis processing speed. It improved RCS values 44.50% on average (P < 0.0002) with actual DNA histograms. Whereas the CS method became less accurate (chi-square test) as the amount of reduction increased, the US method was unaffected, producing consistently better results. The US method is recommended for reducing histogram resolution in modeling applications such as DNA cell-cycle analysis. It may have implications in other areas of flow cytometric data analysis.

Long-term cultivation of colorectal carcinoma cells with anti-cancer drugs induces drug resistance and telomere elongation: an in vitro study.

BackgroundThe role of telomerase activation in the expression and/or maintenance of drug resistance is not clearly understood. Therefore, we investigated the relationships, among the telomerase activity, telomere length and the expression of multidrug resistance genes in colorectal cancer cell lines cultivated with anti-cancer drugs.MethodsLoVo and DLD-1 cells were continuously grown in the presence of both CDDP and 5-FU for up to 100 days. Cell proliferation, telomerase activity, telomere length and the expression of multidrug resistance genes were serially monitored as the PDL increased.ResultsThe expression of multidrug resistance genes tended to increase as the PDL increased. However, an abnormal aneuploid clone was not detected as far as the cells were monitored by a DNA histogram analysis. Tumor cells showing resistance to anti-cancer drugs revealed a higher cell proliferation rate. The telomere length gradually increased with a progressive PDL. The telomerase activity reached a maximum level at 15 PDL in LoVo cells and at 27 PDL in DLD-1 cells. An increase in the mRNA expression of the telomerase components, especially in hTERT and in hTR, was observed at the same PDLs.ConclusionsThese results suggest that a high telomerase activity and an elongation of telomeres both appear to help maintain and/or increase drug resistance in colorectal cancer cells. Cancer cells with long telomeres and a high proliferative activity may thus be able to better survive exposure to anti-cancer drugs. This is presumably due to an increased chromosome stability and a strong expression of both mdr-1 and MRP genes.

Optimizing flow cytometric DNA ploidy and S-phase fraction as independent prognostic markers for node-negative breast cancer specimens.

Developing a reliable and quantitative assessment of the potential virulence of a malignancy has been a long-standing goal in clinical cytometry. DNA histogram analysis provides valuable information on the cycling activity of a tumor population through S-phase estimates; it also identifies nondiploid populations, a possible indicator of genetic instability and subsequent predisposition to metastasis. Because of conflicting studies in the literature, the clinical relevance of both of these potential prognostic markers has been questioned for the management of breast cancer patients. The purposes of this study are to present a set of 10 adjustments derived from a single large study that optimizes the prognostic strength of both DNA ploidy and S-phase and to test the validity of this approach on two other large multicenter studies. Ten adjustments to both DNA ploidy and S-phase were developed from a single node-negative breast cancer database from Baylor College (n = 961 cases). Seven of the adjustments were used to reclassify histograms into low-risk and high-risk ploidy patterns based on aneuploid fraction and DNA index optimum thresholds resulting in prognostic P values changing from little (P < 0.02) or no significance to P < 0.000005. Other databases from Sweden (n = 210 cases) and France (n = 220 cases) demonstrated similar improvement of DNA ploidy prognostic significance, P < 0.02 to P < 0.0009 and P < 0.12 to P < 0.002, respectively. Three other adjustments were applied to diploid and aneuploid S-phases. These adjustments eliminated a spurious correlation between DNA ploidy and S-phase and enabled them to combine independently into a powerful prognostic model capable of stratifying patients into low, intermediate, and high-risk groups (P < 0.000005). When the Baylor prognostic model was applied to the Sweden and French databases, similar significant patient stratifications were observed (P < 0.0003 and P < 0.00001, respectively). The successful transference of the Baylor prognostic model to other studies suggests that the proposed adjustments may play an important role in standardizing this test and provide valuable prognostic information to those involved in the management of breast cancer patients.

Diagnostic accuracy of DNA image cytometry and urinary cytology with cells from voided urine in the detection of bladder cancer

Objectives. To assess the potential of DNA image cytometry in screening for bladder cancer, compare it with conventional urinary cytology, and evaluate its possible use in routine urinary evaluation. Urinary cytology is still the most common method for detection of bladder cancer in routine clinical use. The considerable shortcomings of urinary cytology include its low sensitivity in low-grade carcinomas and its poor reproducibility. Methods. Spontaneously voided urine specimens from 40 patients with grade 1 (n = 27), grade 2 (n = 10), and grade 3 (n = 3) histologically proven transitional cell carcinoma and 40 patients with symptomatic urologic disease of the bladder were analyzed by cytology and DNA image cytometry. The DNA content was determined by use of the CM-1 Cytometer according to the guidelines in the ESACP Consensus Report on Standardization of DNA Image Cytometry. Results. Urinary cytology yielded an overall sensitivity of 47.5%. Conventional analysis of DNA histograms measuring the presence of DNA stemline aneuploidy (1.8c > stemline ploidy [STP] > 2.2c) revealed a sensitivity of 62.5%; applying the stemline interpretation according to Böcking et al. increased the overall sensitivity to 75%. The specificity of both methods was 100%. DNA image cytometry demonstrated a high sensitivity in grade 1 tumors (70.4%) compared with cytology (26%). Conclusions. In light of its highly improved sensitivity compared with urinary cytology, DNA image cytometry should be used to evaluate suspect urothelial cells in urinary cytology specimens. Since the method provides more objective and reproducible results with a specificity comparable to that of cytology, we encourage its primary application in the screening for bladder cancer, provided these results can be confirmed in a multicenter evaluation study.

Kinetic profiles of intraepithelial and invasive prostatic neoplasias: the key role of down-regulated apoptosis in tumor progression.

The cell kinetic of prostatic intraepithelial neoplasia (PIN) is poorly understood. Herein we report the kinetic pattern of PIN, both not associated (primary) and associated (secondary) with coexistent invasive carcinoma (PCa). Surgical specimens collected in 20 cases of primary PIN, 20 of secondary PIN and 20 of PCa were studied by MIB-1 immunostaining, in situ end-labeling (ISEL) and DNA histogram analysis, and the cell density in each case was estimated using the formula N = (n pi/4)2. Fifty high-power fields (HPF), or the complete lesion if smaller, were screened in each lesion, and both mean and standard deviation were recorded. Statistical differences were studied by means of Fisher's exact test. ISEL indices were significantly (P < 0.0001) lower in PCa (0.1 +/- 0.3) than in primary PIN (0.5 +/- 0.3), while the MIB-I indices were similar in both conditions (P = 0.56). Statistically significant differences were also detected for both MIB-1 and ISEL indices when secondary PIN (MIB-1 1.9 +/- 0.7, ISEL 3.7 +/- 3.3) was compared with primary PIN (MIB-1 2.5 +/- 2.1, ISEL 0.5 +/- 0.3) and PCa (P < 0.0001). In terms of cellularity, primary PIN (26.3 +/- 7.1) revealed scores significantly lower (P < 0.0001) than those recorded in PCa (39.0 +/- 8.8) and secondary PIN (32.9 +/- 14.3). In conclusion, early prostatic tumor is mainly defined by down-regulated apoptosis rather than by increased proliferation. Secondary PIN displays unique kinetic features suggesting an evolved stage of primary PIN.