Abnormal DNA Content Research Articles

Tumor aneuploidy correlates with hallmarks of cancer

Abnormalities of cellular DNA content have long been associated with tumorigenesis. Such abnormalities were originally implicated in cancer development over 100 years ago by German biologist Boveri (1), and during the last years it has become clear that cellular aneuploidy is a driving force in the process of carcinogenesis.

Diagnosis and risk stratification of Barrett’s dysplasia by flow cytometric DNA analysis of paraffin-embedded tissue

ObjectiveThe diagnosis of dysplasia in Barrett’s oesophagus (BO) can be challenging, and reliable ancillary techniques are not available. This study examines if DNA content abnormality detected by flow cytometry can...

Assessment of DNA Ploidy in the ProMisE molecular subgroups of endometrial cancer

ObjectiveWe sought to determine whether DNA ploidy correlates with the four molecular subgroups of endometrial carcinoma (EC) as determined using ProMisE (Proactive Molecular Risk Classifier for Endometrial Cancer). Methods90 cases of EC previously characterized by clinicopathological parameters, outcomes, and ProMisE molecular subgroup (POLE EDM, MMR-D, p53 wt or p53 abn) were assessed for DNA ploidy using image cytometry. Associations of ploidy with traditional clinicopathological parameters were also tested. ResultsAbnormal DNA ploidy status differed amongst the ProMisE groups (p<0.001) and was found in 80.9% (17/21) of p53 abn, 37.0% (10/27) of p53 wt, 28.6% (4/14) of POLE EDM and 14.3% (4/28) of MMR-D. Abnormal DNA content was significantly associated with lower BMI (p=0.034) and grade 3 tumors (p=0.001). In the entire cohort, abnormal DNA content was significantly associated with worse progression free survival (p=0.0094) but not disease specific survival (p=0.249) or overall survival (p=0.187). When examining ploidy within each of the ProMisE groups, abnormal DNA content correlated with worse overall survival (p=0.041) and progression free survival (p=0.011) in the MMR-D group. No statistically significant relationship was seen in the remaining 3 groups. ConclusionAbnormal DNA ploidy status did correlate with the molecular subgroups of EC; abnormal DNA content was seen in the large majority of p53 abn cases. Abnormal ploidy however was also seen in smaller numbers in the p53 wt, POLE EDM and MMR-D groups; therefore abnormal DNA content was not a specific marker for any one molecular group. The addition of ploidy to the ProMisE molecular categories conferred additional prognostic value within the MMR-D group, which merits further study.

Meta-analysis of biomarkers predicting risk of malignant progression in Barrett's oesophagus.

Barrett's oesophagus is a precursor to the development of oesophageal adenocarcinoma. This study sought to clarify the role of genetic, chromosomal and proliferation biomarkers that have been the subjects of multiple studies through meta-analysis. MEDLINE, Embase, PubMed and the Cochrane Library were searched for clinical studies assessing the value of p53, p16, Ki-67 and DNA content abnormalities in Barrett's oesophagus. The main outcome measure was the risk of development of high-grade dysplasia (HGD) or oesophageal adenocarcinoma. Some 102 studies, with 12 353 samples, were identified. Mutation (diagnostic odds ratio (DOR) 10·91, sensitivity 47 per cent, specificity 92 per cent, positive likelihood ratio (PLR) 4·71, negative likelihood ratio (NLR) 0·65, area under the curve (AUC) 0·792) and loss (DOR 16·16, sensitivity 31 per cent, specificity 98 per cent, PLR 6·66, NLR 0·41, AUC 0·923) of p53 were found to be superior to the other p53 abnormalities (loss of heterozygosity (LOH) and overexpression). Ki-67 had high sensitivity in identifying high-risk patients (DOR 5·54, sensitivity 82 per cent, specificity 48 per cent, PLR 1·59, NLR 0·42, AUC 0·761). Aneuploidy (DOR 12·08, sensitivity 53 per cent, specificity 87 per cent, PLR 4·26, NLR 0·42, AUC 0·846), tetraploidy (DOR 5·87, sensitivity 46 per cent, specificity 85 per cent, PLR 3·47, NLR 0·65, AUC 0·793) and loss of Y chromosome (DOR 9·23, sensitivity 68 per cent, specificity 80 per cent, PLR 2·67, NLR 0·49, AUC 0·807) also predicted malignant development, but p16 aberrations (hypermethylation, LOH, mutation and loss) failed to demonstrate any advantage over the other biomarkers studied. Loss and mutation of p53, and raised level of Ki-67 predicted malignant progression in Barrett's oesophagus.

Whole Chromosome Instability induces senescence and promotes SASP.

Age-related accumulation of ploidy changes is associated with decreased expression of genes controlling chromosome segregation and cohesin functions. To determine the consequences of whole chromosome instability (W-CIN) we down-regulated the spindle assembly checkpoint component BUB1 and the mitotic cohesin SMC1A, and used four-color-interphase-FISH coupled with BrdU incorporation and analyses of senescence features to reveal the fate of W-CIN cells. We observed significant correlations between levels of not-diploid cells and senescence-associated features (SAFs). W-CIN induced DNA double strand breaks and elevated oxidative stress, but caused low apoptosis. SAFs of W-CIN cells were remarkably similar to those induced by replicative senescence but occurred in only 13 days versus 4 months. Cultures enriched with not-diploid cells acquired a senescence-associated secretory phenotype (SASP) characterized by IL1B, CXCL8, CCL2, TNF, CCL27 and other pro-inflammatory factors including a novel SASP component CLEC11A. These findings suggest that W-CIN triggers premature senescence, presumably to prevent the propagation of cells with an abnormal DNA content. Cells deviating from diploidy have the ability to communicate with their microenvironment by secretion of an array of signaling factors. Our results suggest that aneuploid cells that accumulate during aging in some mammalian tissues potentially contribute to age-related pathologies and inflammation through SASP secretion.

PTU-152 Prediction of Malignant Progression of Barrett’s Oesophagus – A Complete Systematic Review and Meta-Analysis

Introduction Barrett’s Oesophagus (BO) is a precursor for Oesophageal Adenocarcinoma (OAC). Currently, there are no biomarkers in clinical practice that predict the malignant progression in BO. We sought to clarify the effectiveness of common genetic aberrations as potential biomarkers in this context through a systematic review and meta-analyses. Methods MEDLINE, EMBASE and the Cochrane Library were searched by two independent reviewers to identify all clinical studies that assessed efficacy of p53, p16, Ki-67 and DNA content abnormalities as prognostic markers in BO. The main outcome measure was development of OAC. review and meta-analyses. Results 104 clinical studies, with 12,353 samples were identified. Mutation [DOR 10.91 (2.46–48.42), sensitivity 47% (41–53%), specificity 92% (90–94%), PLR 4.71 (1.73–12.78), NLR 0.65 (0.50–0.85), AUC 0.7921] and loss of p53 [DOR 16.16 (6.09–42.88), sensitivity 31% (25–38%), specificity 98% (97–98%), PLR 6.66 (4.51–9.84), NLR 0.41 (0.11–1.47), AUC 0.9228] were found to be superior to the other p53 abnormalities (Loss of heterozygosity and Over-expression). Ki-67 was also noted to have high sensitivity in identifying high risk patients [DOR 5.54 (3.40–9.05), sensitivity 82% (78–85%), specificity 48% (44–52%), PLR 1.59 (1.20–2.10), NLR 0.42 (0.34–0.51), AUC 0.7607]. Aneuploidy [DOR 12.08 (8.09–18.03), sensitivity 53% (50–57%), specificity 87% (85–88%), PLR 4.26 (2.92–6.20), NLR 0.42 (0.32–0.55), AUC 0.8461], tetraploidy [DOR 5.87 (2.56–13.4), sensitivity 46% (39–53%), specificity 85% (82–87%), PLR 3.47 (1.98–6.05), NLR 0.65 (0.45–0.94), AUC 0.7926] and loss of Y chromosome [DOR 9.23 (4.34–19.63), sensitivity 68% (56–79%), specificity 80% (73–86%), PLR 2.67 (1.29–5.50), NLR 0.49 (0.35–0.68), AUC 0.8073] also predicted the malignant development with respectable accuracy but p16 aberrations [{Hypermethylation - DOR 3.00 (2.02–4.45), sensitivity 56% (51–61%), specificity 63% (59–66%), PLR 1.68 (1.35–2.08) (1.32–1.72), NLR 0.66 (0.54–0.80), AUC 0.6773}, {LOH - DOR 4.19 (1.85–9.53), sensitivity 55% (47–62%), specificity 54% (50–58%), PLR 1.84 (1.13–2.99), NLR 0.73 (0.60–0.88), AUC 0.6915}, {Mutation - DOR 1.61 (0.86–3.01), sensitivity 18% (13–24%), specificity 86% (83–89%), PLR 1.53 (0.97–3.42), NLR 0.96 (0.87–1.04), AUC 0.6890}, {Loss - DOR 2.18 (1.18–4.01), sensitivity 58% (53–63%), specificity 58% (53–63%), PLR 1.51 (1.08–1.95), NLR 0.74 (0.56–0.97), AUC 0.6314}] failed to demonstrate any advantage over the other biomarkers studied. Conclusion Loss and mutation of p53 and Ki-67 effectively predict malignant progression in BO. A panel of biomarkers would be more suited to be included in surveillance programme. This will need to be confirmed in large, prospective clinical trials with cost- efficiency analyses. Disclosure of Interest None Declared

The Combination of DNA Ploidy Status and PTEN/6q15 Deletions Provides Strong and Independent Prognostic Information in Prostate Cancer.

Aberrant DNA content has been discussed as a potential prognostic feature in prostate cancer. We analyzed the clinical significance of DNA ploidy in combination with prognostic relevant deletions of PTEN and 6q15 in 3,845 prostate cancers. The DNA status was diploid in 67.8%, tetraploid in 25.6%, and aneuploid in 6.8% of tumors, and deletions of PTEN and 6q15 occurred in 17.8% and 20.3% of tumors. Abnormal DNA content and deletions were linked to high Gleason score, advanced tumor stage, and positive nodal stage (P < 0.0001 each). The risk of PSA recurrence increased from diploid to tetraploid and from tetraploid to aneuploid DNA status (P < 0.0001 each). However, 40% of patients with Gleason score ≥4+4 and 55% of patients with PSA recurrence had diploid cancers. This fraction decreased to 21% (Gleason ≥4+4) and 29% (PSA recurrence) if PTEN and/or 6q deletion data were added to ploidy data to identify cancers with an aberrant DNA status. The significance of combining both deletions and ploidy was further demonstrated in a combined recurrence analysis. Presence of deletions increased the risk of PSA recurrence in diploid (P < 0.0001), tetraploid (P < 0.0001), and aneuploid cancers (P = 0.0049), and the combination of ploidy data and deletions provided clinically relevant information beyond the CAPRA-S nomogram. Multivariate modeling including preoperatively and postoperatively available parameters identified the "combined DNA status" as a strong independent predictor of poor patient outcome. The combinatorial DNA content analysis involving general (ploidy) and specific events (deletions) has the potential for clinical utility in prostate cancer. Clin Cancer Res; 22(11); 2802-11. ©2016 AACR.

Mechanisms and consequences of aneuploidy and chromosome instability in the aging brain

Aneuploidy and polyploidy are a form of Genomic Instability (GIN) known as Chromosomal Instability (CIN) characterized by sporadic abnormalities in chromosome copy numbers. Aneuploidy is commonly linked to pathological states. It is a hallmark of spontaneous abortions and birth defects and it is observed virtually in every human tumor, therefore being generally regarded as detrimental for the development or the maturation of tissues under physiological conditions. Polyploidy however, occurs as part of normal physiological processes during maturation and differentiation of some mammalian cell types. Surprisingly, high levels of aneuploidy are present in the brain, and their frequency increases with age suggesting that the brain is able to maintain its functionality in the presence of high levels of mosaic aneuploidy. Because somatic aneuploidy with age can reach exceptionally high levels, it is likely to have long-term adverse effects in this organ. We describe the mechanisms accountable for an abnormal DNA content with a particular emphasis on the CNS where cell division is limited. Next, we briefly summarize the types of GIN known to date and discuss how they interconnect with CIN. Lastly we highlight how several forms of CIN may contribute to genetic variation, tissue degeneration and disease in the CNS.

Whole slide image cytometry: a novel method to detect abnormal DNA content in Barrett's esophagus

Barrett's esophagus (BE) is a precursor of esophageal adenocarcinoma (EAC). Both low-grade dysplasia (LGD) and high-grade dysplasia (HGD) are associated with an increased risk of progression to EAC. However, histological interpretation and grading of dysplasia (particularly LGD) is subjective and poorly reproducible. This study has combined whole slide imaging with DNA image cytometry to provide a novel method for the detection of abnormal DNA content through image analysis of tissue sections. A total of 20 cases were evaluated, including 8 negative for dysplasia (NFD), 6 LGD, and 6 HGD. Feulgen-stained esophageal sections were scanned in their entirety. Barrett's mucosa was interactively chosen for automatic nuclei segmentation where irrelevant cell types were ignored. The combined DNA content histogram for all nuclei within selected image regions was then obtained. In addition, three histogram measurements were computed, including xER-5C, 2cDI, and DNA-MG. Visual evaluation suggested the shape of DNA content histograms from NFD, LGD, and HGD cases exhibiting identifiable differences. The histogram measurements, xER-5C, 2cDI, and DNA-MG, were shown to be effective in differentiating metaplastic from dysplastic cases with statistical significance. Moreover, they also successfully separated NFD, LGD, and HGD patients with statistical significance. Whole slide image cytometry is a novel and effective method for the detection of abnormal DNA content in BE. Compared with histological review, it is more objective. Compared with flow cytometry and cytology-preparation image cytometry, it is low cost, simple to use, only requires a single 1 μm section, and facilitates selection of tissue and topographical correlation. Whole slide image cytometry can detect differences in DNA content between NFD, LGD, and HGD patients in this cross-sectional study. Abnormal DNA content detection by whole slide image cytometry is a promising biomarker of progression that could affect future diagnostics in BE.

"Indefinite for Dysplasia" in Barrett's Esophagus: Inflammation and DNA Content Abnormality are Significant Predictors of Early Detection of Neoplasia.

Background:Dysplasia arising from Barrett's esophagus precedes esophageal adenocarcinoma (EAC). Cases that are difficult to diagnose as dysplastic, especially in the setting of inflammation, may be designated “indefinite for dysplasia (IND).” Although flow cytometric analysis of DNA content has shown some promise in detecting EAC, there are few reports that have specifically evaluated the outcome of IND.Aims and methods:We analyzed a series of 96 IND patients seen at the University of Washington between 2005 and 2013 to determine the outcome of IND and to identify factors (including histologic features and DNA flow cytometric data) associated with subsequent detection of neoplasia.Results:Twenty-five percent of IND cases were found to have low-grade dysplasia, high-grade dysplasia (HGD), or EAC within 1 year, with 37% and 47% detected within 2 and 3 years, respectively. The 1-, 2-, and 3-year detection rates of HGD or EAC were 10%, 13%, and 20%, respectively. Active inflammation (hazard ratio (HR)=3.4, P=0.0005) and abnormal DNA content (HR=5.7, P=0.003) were significant risk factors of neoplasia. When active inflammation and DNA flow cytometric results were considered together, the HR for the combined markers was 18.8 (P<0.0001). The sensitivity and specificity of the combined markers for predicting detection of subsequent neoplasia within 3 years were 100% and 60%, respectively, with 100% negative and 89% positive predictive values.Conclusions:Histology with the support of DNA flow cytometry can identify a subset of IND patients who may have a higher risk for subsequent detection of neoplasia.

Cellular stress responses in cancer and cancer therapy.

Cellular stress responses in cancer and cancer therapy.

DNA content status using brush biopsy with image cytometry correlated with staging of oral leukoplakia: A preliminary study

Oral leukoplakia (OL) is the best-known potentially malignant disorder of oral cancer. The hypothesis was tested that DNA content abnormality may contribute to risk prediction of malignant potential of OL. All OLs were staged according to a clinicopathologic classification and OL-staging system. DNA content status was investigated in a blinded prospective series of OL using brush biopsy with image cytometry, and examined the correlation of DNA content with the clinicopathologic features and OL-staging system in this preliminary study. Among 65 patients with OL, 27 (41.5%) was identified as DNA content abnormality. The frequency (77.8%) of DNA content abnormality in tongue was higher than that (22.2%) in other oral sites (χ(2) test, P=0.038), and moderate or severe dysplasia had a higher frequency (63.0%) of DNA content abnormality than that (37.0%) of no or mild dysplasia (χ(2) test, P=0.022). Moreover, the odds ratio of DNA content abnormality in high-risk patient group was 5.74-fold (95% confidence interval, 1.81-18.20; P=0.003) increase compared with low-risk patient group. Importantly, the positive correlation between OL-staging system and DNA content status was significant (P=0.018, correlation coefficient=0.292). Our findings showed that DNA content status correlated with OL-staging system, suggesting that DNA content abnormality in OL as detected by image cytometry was an early event in oral carcinogenesis. The further large-scale prospective studies with clinical endpoints are warranted to validate the value of DNA image cytometry.

Toxicity of drinking water disinfection byproducts: cell cycle alterations induced by the monohaloacetonitriles.

Haloacetonitriles (HANs) are a chemical class of drinking water disinfection byproducts (DBPs) that form from reactions between disinfectants and nitrogen-containing precursors, the latter more prevalent in water sources impacted by algae bloom and municipal wastewater effluent discharge. HANs, previously demonstrated to be genotoxic, were investigated for their effects on the mammalian cell cycle. Treating Chinese hamster ovary (CHO) cells with monoHANs followed by the release from the chemical treatment resulted in the accumulation of abnormally high DNA content in cells over time (hyperploid). The potency for the cell cycle alteration followed the order: iodoacetonitrile (IAN) > bromoacetonitrile (BAN) ≫ chloroacetonitrile (CAN). Exposure to 6 μM IAN, 12 μM BAN and 900 μM CAN after 26 h post-treatment incubation resulted in DNA repair; however, subsequent cell cycle alteration effects were observed. Cell proliferation of HAN-treated cells was suppressed for as long as 43 to 52 h. Enlarged cell size was observed after 52 h post-treatment incubation without the induction of cytotoxicity. The HAN-mediated cell cycle alteration was mitosis- and proliferation-dependent, which suggests that HAN treatment induced mitosis override, and that HAN-treated cells proceeded into S phase and directly into the next cell cycle. Cells with multiples genomes would result in aneuploidy (state of abnormal chromosome number and DNA content) at the next mitosis since extra centrosomes could compromise the assembly of bipolar spindles. There is accumulating evidence of a transient tetraploid state proceeding to aneuploidy in cancer progression. Biological self-defense systems to ensure genomic stability and to eliminate tetraploid cells exist in eukaryotic cells. A key tumor suppressor gene, p53, is oftentimes mutated in various types of human cancer. It is possible that HAN disruption of the normal cell cycle and the generation of aberrant cells with an abnormal number of chromosomes may contribute to cancer induction and perhaps be involved in the induction of adverse pregnancy outcomes associated with long-term consumption of disinfected water. Here we present the first observation of the induction of hyperploidy by a class of DBPs.

Metabolic Complications of in utero Maternal HIV and Antiretroviral Exposure in HIV-exposed Infants

Despite a wide body of literature supporting the use of antenatal antiretrovirals (ARV) for the prevention of mother-to-child transmission, there remains a need for continued monitoring as the intrauterine interval is a critical period during which fetal programming influences the future health and development of the child. We conducted a systematic review of the current literature addressing potential metabolic complications of in utero HIV and ARV exposure. We describe studies evaluating metabolic outcomes such as intrauterine and early postnatal growth, bone health and mitochondrial toxicity. Overall, infants exposed to HIV/ARV do not appear to exhibit vastly compromised intrauterine or early postnatal growth. However, some studies on the effect of combination antiretroviral therapy on small for gestational age and low birth weight outcomes in low-middle income countries show a risk for small for gestational age/low birth weight while those in the United States do not. Postnatal growth to 1 year does not appear to be affected by intrauterine tenofovir exposure in African studies, but a US study found statistically significant differences in length for age z scores (LAZ) at 1 year. Little data exists on long-term bone health. Mitochondrial toxicity including abnormal mitochondrial morphology and DNA content, as well as neurologic deficits and death, have been demonstrated in HIV/ARV-exposed infants. Although gross measures of metabolic well-being appear to be reassuring, careful vigilance of even small risks for potential serious adverse effects to infants exposed to intrauterine HIV/ARVs is warranted as intrauterine fetal metabolic programming may substantially impact the future health of the child.

Adaptation of image cytometry methodology for DNA ploidy analysis of cervical epithelium samples: A pilot study

ObjectiveTo determine DNA ploidy in the cervical specimens of patients revealing a suspicion of cancer by image analysis performed by using a combination of commercial analysis software, conventional microscopy, and certified filters. Materials and methodsThis study followed a prospective design. Cervical samples were obtained from 20 patients undergoing routine screening in the Gynecologic-Oncology Unit of the University Hospital of the Federal University of Minas Gerais, Brazil. Three slides were prepared for each case and the DNA content was determined by image cytometry, post Feulgen staining. DNA ploidy, as well as events exceeding 5C and 9C, was assessed according to the guidelines and algorithms prescribed for diagnostic interpretation by the European Society for Analytical Cellular Pathology. ResultsBy employing the adapted tool, identification of the lesions with euploid and aneuploid profiles was possible. Abnormal DNA content was found in 65% of the cases (13/20), with 45% (9/20) presenting nuclei with >5C content and 20% (4/20) with >9C content. In the analyses conducted in this study, the coefficient of variation with respect to DNA quantity was lower than the 5% threshold recommended by the European Society for Analytical Cellular Pathology. ConclusionImage cytometry of the cervical specimens revealed DNA aneuploidy, most probably resulting from chromosomal alterations and appearing as precancerous lesions in 65% of the cases. The adaptations implemented in this study, enabled the DNA-image cytometry to become more accessible, enhancing its extended use as an adjuvant strategy for the early screening of the cervical epithelium samples during routine analyses.

Genetic Validation of Aminoacyl-tRNA Synthetases as Drug Targets in Trypanosoma brucei

Human African trypanosomiasis (HAT) is an important public health threat in sub-Saharan Africa. Current drugs are unsatisfactory, and new drugs are being sought. Few validated enzyme targets are available to support drug discovery efforts, so our goal was to obtain essentiality data on genes with proven utility as drug targets. Aminoacyl-tRNA synthetases (aaRSs) are known drug targets for bacterial and fungal pathogens and are required for protein synthesis. Here we survey the essentiality of eight Trypanosoma brucei aaRSs by RNA interference (RNAi) gene expression knockdown, covering an enzyme from each major aaRS class: valyl-tRNA synthetase (ValRS) (class Ia), tryptophanyl-tRNA synthetase (TrpRS-1) (class Ib), arginyl-tRNA synthetase (ArgRS) (class Ic), glutamyl-tRNA synthetase (GluRS) (class 1c), threonyl-tRNA synthetase (ThrRS) (class IIa), asparaginyl-tRNA synthetase (AsnRS) (class IIb), and phenylalanyl-tRNA synthetase (α and β) (PheRS) (class IIc). Knockdown of mRNA encoding these enzymes in T. brucei mammalian stage parasites showed that all were essential for parasite growth and survival in vitro. The reduced expression resulted in growth, morphological, cell cycle, and DNA content abnormalities. ThrRS was characterized in greater detail, showing that the purified recombinant enzyme displayed ThrRS activity and that the protein localized to both the cytosol and mitochondrion. Borrelidin, a known inhibitor of ThrRS, was an inhibitor of T. brucei ThrRS and showed antitrypanosomal activity. The data show that aaRSs are essential for T. brucei survival and are likely to be excellent targets for drug discovery efforts.

Proteins related to early changes in carcinogenesis of hepatic oval cells after treatment with methylnitronitrosoguanidine

Hepatic oval cells are considered as facultative progenitor/stem cells of liver and able to differentiate into either hepatocytes or biliary epithelial cells. The transformed oval cells by carcinogen possess potential to develop carcinomas in animal models. In order to better understand the molecular mechanism in carcinogenetic process, we used a proteomic approach to assess the early changes in protein expression of oval cells (OC3W3-15) initiated by methylnitronitrosoguanidine (MNNG). Meanwhile, we compared cell biologic characteristics of the MNNG treated OC3W3-15 cells and control oval cells by electron microscopy, flow cytometry, karyotype and soft agar assay. The mRNA levels of GGT and GSTP1 determined by real-time PCR were also detected in both cell lines. Our results showed that MNNG-treated OC3W3-15 cells exhibited characteristics of malignant transformation, including growth rate, chromosomal aberrations, abnormal DNA content, and the ability to form colonies. The cells expressed higher levels of the tumor marker AFP, GGT and GSTP1 mRNA than that of control cells. Significant changes of several proteins involved in the malignant transformation process, including cell cycle related proteins, proteins involved in organism development and cell differentiation, are found in OC3W3-15 cells. The proteins may provide early affection in malignant transformation of hepatic oval cells, and yield further insight into mechanism of carcinogenesis of hepatocellular carcinoma.

Individual karyotypes at the origins of cervical carcinomas

BackgroundIn 1952 Papanicolaou et al. first diagnosed and graded cervical carcinomas based on individual “abnormal DNA contents” and cellular phenotypes. Surprisingly current papilloma virus and mutation theories of carcinomas do not mention these individualities. The viral theory holds that randomly integrated, defective genomes of papilloma viruses, which are often untranscribed, cause cervical carcinomas with unknown cofactors 20–50 years after infection. Virus-free carcinomas are attributed to mutations of a few tumor-suppressor genes, especially the p53 gene. But the paradox of how a few mutations or latent defective viral DNAs would generate carcinomas with endless individual DNA contents, degrees of malignancies and cellular phenotypes is unsolved. Since speciation predicts individuality, we test here the theory that cancers are autonomous species with individual clonal karyotypes and phenotypes. This theory postulates that carcinogens induce aneuploidy. By unbalancing mitosis genes aneuploidy catalyzes chain reactions of karyotypic evolutions. Most such evolutions end with non-viable karyotypes but a few become new cancer karyotypes. Despite congenitally unbalanced mitosis genes cancer karyotypes are stabilized by clonal selections for cancer-specific autonomy.ResultsTo test the prediction of the speciation theory that individual carcinomas have individual clonal karyotypes and phenotypes, we have analyzed here the phenotypes and karyotypes of nine cervical carcinomas. Seven of these contained papilloma virus sequences and two did not. We determined phenotypic individuality and clonality based on the morphology and sociology of carcinoma cells in vitro. Karyotypic individuality and clonality were determined by comparing all chromosomes of 20 karyotypes of carcinomas in three-dimensional arrays. Such arrays list chromosome numbers on the x-axis, chromosome copy numbers on the y-axis and the number of karyotypes arrayed on the z-axis. We found (1) individual clonal karyotypes and phenotypes in all nine carcinomas, but no virus-specific markers, (2) 1-to-1 variations between carcinoma-specific karyotypes and phenotypes, e.g. drug-resistance and cell morphology, (3) proportionality between the copy numbers of chromosomes and the copy numbers of hundreds of over- and under-expressed mRNAs, (4) evidence that tobacco-carcinogens induce cervical carcinomas via aneuploidy, consistent with the speciation theory.ConclusionsSince the individual clonal karyotypes of nine carcinomas correlated and co-varied 1-to-1 with complex individual transcriptomes and phenotypes, we have classical genetic and functional transcriptomic evidence to conclude that these karyotypes encode carcinomas - much like the clonal karyotypes that encode conventional species. These individual karyotypes explain the individual “DNA contents”, the endless grades of malignancies and the complex individual transcriptomes and phenotypes of carcinomas.

Abstract 709: Mitosis interference of cancer cells by NovoTTF-100A causes decreased cellular viability.

Abstract Cancer cell death from traditional cytotoxic chemotherapy and radiation is a result of triggering cell cycle checkpoint with p53 playing a prominent role in orchestrating this effect. Unlike these traditional approaches, the NovoTTF-100A device, which has been approved for the treatment of recurrent glioblastomas, kills tumor cells by a novel mechanism that interferes with the proper progression through mitosis. As is the case with earlier therapeutic interventions, only a subset of patients shows significant response to TTFields treatment. Our research reveals that the tumor-treating fields (TTFields) affects processes proximal to the metaphase-to-anaphase transition, leading to cell cycle arrest in anaphase and eventual cell death. Tissue culture cells exposed to the TTFields during mitosis exhibit a variety of outcomes, including cell arrest in anaphase, cytokinesis with cytokinetic furrow regression, and slippage out of mitosis without division. Time-lapse movies of cells stained with the DNA stain DRAQ5 and exposed to the TTFields revealed membrane blebbing and oscillation, as well as failed segregation of sister chromatids from the metaphase plate, both of which were initiated near the time of metaphase-to-anaphase transition. Immunoflourescence of cells after short-term treatment with TTFields during mitosis showed disordered separation of chromosomes from metaphase plates accompanied by lagging chromosomes, dispersion of chromosomes from the metaphase plate, chromosomes de-condensation in the absence of cytokinesis, and daughter cells with asymmetric chromosomal segregation. Flow cytometry also revealed the presence of cells with abnormal DNA content in cultures treated with TTFields. In contrast to the p53 mutant cell lines, MDA-MB-231 and HCT 116, U87 cells with wild-type p53 exhibit increased Annexin V binding, Caspase activation and increased markers of endoplasmic reticulum (ER) stress after brief exposure during mitosis. This suggests the TTFields induced cellular damage is able to trigger p53-dependent cell death in cells that escape killing during M-phase. These data show that the effects of TTFields on cellular damage during M-phase and provide evidence for the notion that the differential responses to treatment seen in patients may be rooted in the underlying genetic alterations in their tumors. Citation Format: Sze Xian Lee, Eric T. Wong, Kenneth D. Swanson. Mitosis interference of cancer cells by NovoTTF-100A causes decreased cellular viability. [abstract]. In: Proceedings of the 104th Annual Meeting of the American Association for Cancer Research; 2013 Apr 6-10; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2013;73(8 Suppl):Abstract nr 709. doi:10.1158/1538-7445.AM2013-709

High incidence of DNA ploidy abnormalities and increased Mcm2 expression may predict malignant change in oral proliferative verrucous leukoplakia

To assess the DNA content of cases of oral proliferative verrucous leukoplakia (PVL) and correlate the DNA ploidy findings with the expression of Mcm2, geminin, and Ki67, and with clinicopathological data. DNA quantification was performed by image cytometry using the ACIS III Automated Cellular Imaging System. Expression of Ki67, Mcm2 and geminin was determined by immunohistochemistry. There were 21 cases of PVL, the female/male ratio was 6:1, and the average age was 65.5years. Seventeen patients (81.0%) did not report tobacco and alcohol consumption. Nine patients (42.9%) developed verrucous or squamous cell carcinoma. Levels of Mcm2 expression showed a positive correlation with increasingly severe epithelial changes (P=0.03). Twenty patients had their DNA examined by ACIS III, and 19 (95%) showed aneuploidy. The frequency and severity of aneuploidy (P<0.0001), the mean values of the DNA heterogeneity index (P<0.0001) and the 5n-exceeding fractions (P= 0.0007) increased according to epithelial alterations. Abnormal DNA content was observed even in the more indolent lesions. Mcm2 expression and DNA ploidy analysis could be used to predict areas of malignant transformation. The clinicopathological findings associated with the immunohistochemical and DNA ploidy results support the distinct and aggressive profile of this entity.