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Background. For the proper diagnosis and optimal prognosis of acute leukemia (AL), a complex of clinical and laboratory methods is used. Among them, chromosomal analysis plays an important role. Cytogenetic markers of leukemic cells are widely used in clinical settings. WHO classification of Tumors of Haematopoietic and Lymphoid Tissues is based on a combined analysis of clinical, cytomorphological, immunophenotypic and cytogenetic findings. Immunophenotyping of blast cells using monoclonal antibodies has significantly improved the diagnosis of leukemia by detecting the grade of tumor differentiation. Aim: based on the results of immunophenotypic and cytogenetic examinations, to describe the structure of AL and to study the effect of cytogenetic abnormalities on the prognosis of acute leukemia in the child population of the Arkhangelsk region. Material and Methods. Archived data were used to analyze the results of cytogenetic and immunophenotypic examinations of all children aged 0–17 years in the Arkhangelsk region, who were diagnosed with AL in the period from January, 2004 to december, 2018. Results. Immunophenotypic examination of bone marrow blast cells in patients with AL revealed that B-linear lymphoblastic leukemia was the most common (74.8 %). Among the quantitative anomalies, hyperploidy was the most common (additional chromosomes 4, 10 and 17 were observed). The t(12; 21) was the most frequent structural chromosomal abnormality. The philadelphia chromosome (ph) (9;22) was found in 6.5 % of cases. The highest risk of death was observed in AL patients with the acute myeloid leukemia phenotype (p

Hepatitis B virus (HBV) is an aetiological factor for liver cirrhosis and hepatocellular carcinoma (HCC). Despite current antiviral therapies that successfully reduce the viral load in patients with chronic hepatitis B, persistent hepatitis B surface antigen (HBsAg) remains a risk factor for HCC. To explore whether intrahepatic viral antigens contribute directly to hepatocarcinogenesis, we monitored the mitotic progression of HBV-positive cells. Cytokinesis failure was increased in HBV-positive HepG2.2.15 and 1.3ES2 cells, as well as in HuH-7 cells transfected with a wild-type or X-deficient HBV construct, but not in cells transfected with an HBsAg-deficient construct. We show that expression of viral large surface antigen (LHBS) was sufficient to induce cytokinesis failure of immortalized hepatocytes. Premitotic defects with DNA damage and G2 /M checkpoint attenuation preceded cytokinesis in LHBS-positive cells, and ultimately resulted in hyperploidy. Inhibition of polo-like kinase-1 (Plk1) not only restored the G2 /M checkpoint in these cells, but also suppressed LHBS-mediated in vivo tumourigenesis. Finally, a positive correlation between intrahepatic LHBS expression and hepatocyte hyperploidy was detected in >70% of patients with chronic hepatitis B. We conclude that HBV LHBS provokes hyperploidy by inducing DNA damage and upregulation of Plk1; the former results in atypical chromatin structures, and the latter attenuates the function of the G2 /M DNA damage checkpoint. Our data uncover a mechanism by which genomic integrity of hepatocytes is disrupted by viral LHBS. These findings highlight the role of intrahepatic surface antigen as an oncogenic risk factor in the development of HCC. Copyright © 2018 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.

Aims The existence of malignant mesothelioma in situ (MIS) is often postulated, but there are no accepted morphological criteria for making such a diagnosis. Methods and results Here we report two cases that appear to be true MIS on the basis of in-situ genomic analysis. In one case the patient had repeated unexplained pleural unilateral effusions. Two thoracoscopies 9 months apart revealed only visually normal pleura. Biopsies from both thoracoscopies showed only a single layer of mildly reactive mesothelial cells. However, these cells had lost BRCA1-associated protein 1 (BAP1) and showed loss of cyclin-dependent kinase inhibitor 2 (CDKN2A) (p16) by fluorescence in-situ hybridisation (FISH). NF2 was not deleted by FISH but 28% of the mesothelial cells showed hyperploidy. Six months after the second biopsy the patient has persisting effusions but no evidence of pleural malignancy on imaging. The second patient presented with ascites and minimal omental thickening on imaging, but no visual evidence of tumour at laparoscopy. Omental biopsy showed a single layer of minimally atypical mesothelial cells with rare tiny foci of superficial invasion of fat. BAP1 immunostain showed loss of nuclear BAP1 in all the surface mesothelial cells and the invasive cells. There was CDKN2A deletion, but no deletion of NF2 by FISH. Conclusions These cases show that morphologically bland single-layered surface mesothelial proliferations with molecular alterations seen previously only in invasive malignant mesotheliomas exist, and presumably represent malignant MIS. More cases are need to understand the frequency of such changes and the time-course over which invasive tumour develops.

Cancer cells are subjected to constant selection by the immune system, meaning that tumors that become clinically manifest have managed to subvert or hide from immunosurveillance. Immune control can be facilitated by induction of autophagy, as well as by polyploidization of cancer cells. While autophagy causes the release of ATP, a chemotactic signal for myeloid cells, polyploidization can trigger endoplasmic reticulum stress with consequent exposure of the "eat-me" signal calreticulin on the cell surface, thereby facilitating the transfer of tumor antigens into dendritic cells. Hence, both autophagy and polyploidization cause the emission of adjuvant signals that ultimately elicit immune control by CD8+ T lymphocytes. We investigated the possibility that autophagy and polyploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. Mass spectrometry led to the identification of peptides that were presented on major histocompatibility complex (MHC) class I molecules in an autophagy-dependent fashion or that were specifically exposed on the surface of polyploid cells, yet lost upon passage of such cells through immunocompetent (but not immunodeficient) mice. However, the preferential recognition of autophagy-competent and polyploid cells by the innate and cellular immune systems did not correlate with the preferential recognition of such peptides in vivo. Moreover, vaccination with such peptides was unable to elicit tumor growth-inhibitory responses in vivo. We conclude that autophagy and polyploidy increase the immunogenicity of cancer cells mostly by affecting their adjuvanticity rather than their antigenicity.

Osteosarcoma (OS) is the most common primary malignant bone tumor in children and adolescents. It is characterized by highly complex karyotypes with structural and numerical chromosomal alterations. The observed OS-specific characteristics in localization and frequencies of chromosomal breakages strongly implicate a specific set of responsible driver genes or a specific mechanism of fragility induction. In this study, a comprehensive assessment of somatic copy number alterations (SCNAs) was performed in 160 OS samples using whole-genome CytoScan High Density arrays (Affymetrix, Santa Clara, CA). Genes or regions frequently targeted by SCNAs were identified. Breakage analysis revealed OS specific unstable regions in which well-known OS tumor suppressor genes, including TP53, RB1, WWOX, DLG2 and LSAMP are located. Certain genomic features, such as transposable elements and non-B DNA-forming motifs were found to be significantly enriched in the vicinity of chromosomal breakage sites. A complex breakage pattern-chromothripsis-has been suggested as a widespread phenomenon in OS. It was further demonstrated that hyperploidy and in particular chromothripsis were strongly correlated with OS patient clinical outcome. The revealed OS-specific fragility pattern provides novel clues for understanding the biology of OS.

MotivationHyperploidy and segmental aneuploidy are hallmarks of cancer cells due to chromosome segregation errors and genomic instability. In such situations, accurate aneuploidy profiling of cancer data is critical for calibration of copy number (CN)-detection tools. Additionally, cancer cell populations suffer from different levels of clonal heterogeneity and aneuploidy alterations over time. The degree of heterogeneity adversely affects the segregation of the depth of coverage (DOC) signal into integral CN states. This, in turn, strongly influences the reliability of this data for ploidy profiling and copy number variation (CNV) analysis.ResultsWe developed AStra framework for aneuploidy profiling of cancer data and assessing their suitability for copy number analysis without any prior knowledge of the input sequencing data. AStra estimates the best-fit aneuploidy profile as the spectrum with most genomic segments around integral CN states. We employ this spectrum to extract the CN-associated features such as the homogeneity score (HS), whole-genome ploidy level, and CN correction factor. The HS measures the percentage of genomic regions around CN states. It is used as a reliability assessment of sequencing data for downstream aneuploidy profiling and CNV analysis. We evaluated the accuracy of AStra using 31 low-coverage datasets from 20 cancer cell lines. AStra successfully identified the aneuploidy spectrum of complex cell lines with HS greater than 75%. Benchmarking against nQuire tool showed that AStra is superior in detecting the ploidy level using both low- and high-coverage data. Furthermore, AStra accurately estimated the ploidy of 26/27 strains of MCF7 (hyperploid) cell line which exhibit varied levels of aneuploidy spectrum and heterogeneity. Remarkably, we found that HS is strongly correlated with the doubling time of these strains.Availability and implementationAStra is an open source software implemented in Python and is available at https://github.com/AISKhalil/AStra

We summarize the results and clinical usefulness of cytogenetic analysis that is routinely performed for musculoskeletal tumors. We performed cytogenetic analysis and traditional histologic evaluation on 101 (51 malignant/ 50 benign) consecutive tumors that were surgically excised. The successful culture rate for cytogenetic analysis was 86% (87/101). Fifty-four percent (25/46) of clearly malignant tumors that were successfully cultured demonstrated significant clonal abnormalities. Fifty-one percent (21/41) of benign tumors that were cultured had significant cytogenetic clonal aberrations. Increased cellular ploidy (> 50 chromosomes/cell) was demonstrated in 14/46 malignant and 1/41 benign tumors that were successfully cultured. Hyperploidy was highly correlated with malignancy (p < 0.001); the only "benign" tumor was a multiply recurrent and giant cell, demonstrating histologic changes consistent with early sarcomatous transformation. As expected, cytogenetic abnormalities frequently occurred in malignant tumors. Surprisingly, almost half of the benign tumors had significant clonal cytogenetic aberrations. Consistent findings of extra chromosomes 5 and 7 in samples of pigmented villonodular synovitis strongly favored a neoplastic origin for this condition. Although the presence or absence of cytogenetic aberrations cannot be used to determine malignant potential, increased cellular ploidy is highly indicative of malignancy. Modern molecular genetics have become more popular, but cytogenetic analysis can be useful for classifying the malignant potential of recurrent and difficult to diagnose tumors of the musculoskeletal system.

Cell cycle reentry followed by neuronal hyperploidy and synaptic failure are two early hallmarks of Alzheimer’s disease (AD), however their functional connection remains unexplored. To address this question, we induced cell cycle reentry in cultured cortical neurons by expressing SV40 large T antigen. Cell cycle reentry was followed by hyperploidy in ~70% of cortical neurons, and led to progressive axon initial segment loss and reduced density of dendritic PSD-95 puncta, which correlated with diminished spike generation and reduced spontaneous synaptic activity. This manipulation also resulted in delayed cell death, as previously observed in AD-affected hyperploid neurons. Membrane depolarization by high extracellular potassium maintained PSD-95 puncta density and partially rescued both spontaneous synaptic activity and cell death, while spike generation remained blocked. This suggests that AD-associated hyperploid neurons can be sustained in vivo if integrated in active neuronal circuits whilst promoting synaptic dysfunction. Thus, cell cycle reentry might contribute to cognitive impairment in early stages of AD and neuronal death susceptibility at late stages., This work was supported by Ministerio de Economía y Competitividad Grants SAF2015-68488-R (J.M.F.) and BFU2013-47265-R and BFU2016-75107-P (G.P.), and Intramural Grant 201620I017 (G.P.).

The immune system avoids oncogenesis and slows down tumor progression through a mechanism called immunosurveillance. Nevertheless, some malignant cells manage to escape from immune control and form clinically detectable tumors. Tetraploidy, which consists in the intrinsically unstable duplication of the genome, is considered as a (pre)-cancerous event that can result in aneuploidy and contribute to oncogenesis. We previously described the fact that tetraploid cells can be eliminated by the immune system. Here, we investigate the role of different innate and acquired immune effectors by inoculating hyperploid cancer cells into wild type or mice bearing different immunodeficient genotypes (Cd1d(−/−), FcRn(−/−), Flt3l(−/−), Foxn1(nu/nu), MyD88(−/−), Nlrp3(−)(/)(−), Ighm(tm1Cgn), Rag2(−/−)), followed by the monitoring of tumor incidence, growth and final ploidy status. Our results suggest that multiple different immune effectors including B, NK, NKT and T cells, as well as innate immune responses involving the interleukine-1 receptor and the Toll-like receptor systems participate to the immunoselection against hyperploid cells. Hence, optimal anticancer immunosurveillance likely involves the contribution of multiple arms of the immune system.

Numerical chromosomal abnormalities are the leading cause of pregnancy loss and developmental disabilities in humans. There are very few numerical chromosomal abnormalities which affect male reproduction and Klinefelter syndrome accounts for the vast majority of these cases. In addition to the reproductive morbidity associated with Klinefelter syndrome, there are several noteworthy non-reproductive sequelae associated with it. Live births are achievable in Klinefelter men via testicular extraction of sperm and in vitro fertilization with intracytoplasmic sperm injection. Preimplantation genetic screening is also available for these patients as there have been reports of a higher incidence of sex chromosome hyperploidy and autosomal aneuploidies in the sperm of men with Klinefelter syndrome. These patients however need to be counseled about the current limitations of this technology.

OBJECTIVEThere are currently no reliable means to predict the wide variability in behavior of clival chordoma so as to guide clinical decision-making and patient education. Furthermore, there is no method of predicting a tumor’s response to radiation therapy.METHODSA molecular prognostication panel, consisting of fluorescence in situ hybridization (FISH) of the chromosomal loci 1p36 and 9p21, as well as immunohistochemistry for Ki-67, was prospectively evaluated in 105 clival chordoma samples from November 2007 to April 2016. The results were correlated with overall progression-free survival after surgery (PFSS), as well as progression-free survival after radiotherapy (PFSR).RESULTSAlthough Ki-67 and the percentages of tumor cells with 1q25 hyperploidy, 1p36 deletions, and homozygous 9p21 deletions were all found to be predictive of PFSS and PFSR in univariate analyses, only 1p36 deletions and homozygous 9p21 deletions were shown to be independently predictive in a multivariate analysis. Using a prognostication calculator formulated by a separate multivariate Cox model, two 1p36 deletion strata (0%–15% and > 15% deleted tumor cells) and three 9p21 homozygous deletion strata (0%–3%, 4%–24%, and ≥ 25% deleted tumor cells) accounted for a range of cumulative hazard ratios of 1 to 56.1 for PFSS and 1 to 75.6 for PFSR.CONCLUSIONSHomozygous 9p21 deletions and 1p36 deletions are independent prognostic factors in clival chordoma and can account for a wide spectrum of overall PFSS and PFSR. This panel can be used to guide management after resection of clival chordomas.

An at least transient increase of ploidy, usually by whole genome duplication, is a frequent event in oncogenesis. Hyperploidization triggers an endoplasmic reticulum (ER) stress characterized by the hyperphosphorylation of the eukaryotic initiation factor 2α (eIF2α), with leads to the exposure of calreticulin to the cell surface. Calreticulin is acting like an "adjuvant" effect as a "eat me" signal, for dendritic cells, ultimately eliciting immune control by CD8 + T lymphocytes. We investigated the possibility that hyperploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. However, vaccination with candidate peptides was unable to elicit tumor growth-inhibitory responses against hyperploid cells in vivo. We conclude that hyperploidy increases the immunogenicity of cancer cells mostly through their adjuvanticity (through calreticulin exposure) rather than their antigenicity. We also show that when developed in Rag2 - / - γc - / - immunodeficient mice (but not in WT mice) fibrosarcomas induced by methylcholanthrene (MCA) are particularly hyperploid, correlating with higher DNA content, increased nuclear surface, as well as the hyperphosphorylation of eIF2α. These cells are able to form tumors in Rag2 - / - γc - / - recipients (which lack T, B and NK cells) as well as in Rag2 - / - recipients (which only lack T and B lymphocytes) without any difference between the two strains, suggesting that the absence of B and T cells is enough to allow these cells to grow. To measure these parameters, we developed a morphometric analysis tool that is applicable to immunohistochemistry. This software automatically identifies and quantifies the surface of nuclei and the intensity of eIF2α phosphorylation within a perinuclear region of interest. Comparative analyzes validated the accuracy of this method, which can be used to investigate ploidy and ER stress in cancers in situ.; Une augmentation au moins transitoire de la ploïdie est un événement fréquent de l'oncogenèse. L'hyperploïdisation déclenche un stress du réticulum endoplasmique (RE) caractérisé par l'hyperphosphorylation du facteur d'initiation eucaryote 2α (eIF2α) qui induit l'exposition consécutive de la calréticuline à la surface cellulaire. La calréticuline exerce un effet « adjuvant » en tant que un signal "mange-moi", pour les cellules dendritiques, entrainant un contrôle immunitaire de la tumeur par les lymphocytes T CD8+. Nous avons étudié la possibilité que l'hyperploïdisation puisse également affecter l'antigénicité des cellules cancéreuses en modifiant l'immunopeptidome. Cependant, la vaccination avec des peptides candidats n’a pas réussi à induire des réponses inhibitrices de la croissance tumorale de cellules hyperploïdes in vivo. Nous concluons donc que l'hyperploïdie augmente principalement l'immunogénicité des cellules cancéreuses en affectant leur adjuvanticité (via l’expression de calréticuline) plutôt que leur antigénicité. Nous montrons de plus que des fibrosarcomes induits par méthylcholanthrène (MCA), développés dans des souris Rag2-/-γc-/- immunodéficientes (mais pas dans des souris sauvages), sont particulièrement hyperploïdes, en corrélation avec une teneur en ADN plus élevée, une augmentation de la surface nucléaire, ainsi que l'hyperphosphorylation de eIF2α,. De telles cellules forment des tumeurs dans les souris Rag2-/-γc-/- (dépourvues de cellules T, B et NK) ainsi que dans les souris Rag2-/- (dépourvues de lymphocytes T et B) sans différence entre les deux souches de souris, suggérant que l'absence de cellules B et T est suffisante pour que ces cellules prolifèrent. Pour mesurer ces paramètres, nous avons développé un outil d'analyse morphométrique applicable à l'immunohistochimie. Cet algorithme identifie et quantifie automatiquement la surface des noyaux et l'intensité de la phosphorylation de eIF2α dans une région d'intérêt périnucléaire. Des analyses comparatives ont validé la précision de cette méthode, qui peut être utilisée pour étudier la ploïdie et le stress du RE dans les cancers in situ.

Disinfection by-products occur widely as the unintended effect of water disinfection and are associated with toxicity and adverse human health effects. Yet the molecular mechanisms of their toxicity are not well understood. To investigate the molecular basis of hyperploidy induction by monohaloacetonitriles, the interaction of monohaloacetonitriles with topoisomerase II in Chinese hamster ovary cells was examined. We showed a concentration-dependent inhibition of DNA decatenation activity of topoisomerase under acellular conditions while in vitro monohaloacetonitrile treatment expressed mixed results. The working hypothesis, that topoisomerase II is a molecular target of monohaloacetonitriles, was only partially supported. Nevertheless, this research serves as a starting point toward molecular mechanisms of toxic action of monohaloacetonitriles.

An at least transient increase of ploidy, usually by whole genome duplication, is a frequent event in oncogenesis. Hyperploidization triggers an endoplasmic reticulum (ER) stress characterized by the hyperphosphorylation of the eukaryotic initiation factor 2α (eIF2α), with leads to the exposure of calreticulin to the cell surface. Calreticulin is acting like an "adjuvant" effect as a "eat me" signal, for dendritic cells, ultimately eliciting immune control by CD8 + T lymphocytes. We investigated the possibility that hyperploidization might also affect the antigenicity of cancer cells by altering the immunopeptidome. However, vaccination with candidate peptides was unable to elicit tumor growth-inhibitory responses against hyperploid cells in vivo. We conclude that hyperploidy increases the immunogenicity of cancer cells mostly through their adjuvanticity (through calreticulin exposure) rather than their antigenicity. We also show that when developed in Rag2 - / - γc - / - immunodeficient mice (but not in WT mice) fibrosarcomas induced by methylcholanthrene (MCA) are particularly hyperploid, correlating with higher DNA content, increased nuclear surface, as well as the hyperphosphorylation of eIF2α. These cells are able to form tumors in Rag2 - / - γc - / - recipients (which lack T, B and NK cells) as well as in Rag2 - / - recipients (which only lack T and B lymphocytes) without any difference between the two strains, suggesting that the absence of B and T cells is enough to allow these cells to grow. To measure these parameters, we developed a morphometric analysis tool that is applicable to immunohistochemistry. This software automatically identifies and quantifies the surface of nuclei and the intensity of eIF2α phosphorylation within a perinuclear region of interest. Comparative analyzes validated the accuracy of this method, which can be used to investigate ploidy and ER stress in cancers in situ., Une augmentation au moins transitoire de la ploïdie est un événement fréquent de l'oncogenèse. L'hyperploïdisation déclenche un stress du réticulum endoplasmique (RE) caractérisé par l'hyperphosphorylation du facteur d'initiation eucaryote 2α (eIF2α) qui induit l'exposition consécutive de la calréticuline à la surface cellulaire. La calréticuline exerce un effet « adjuvant » en tant que un signal "mange-moi", pour les cellules dendritiques, entrainant un contrôle immunitaire de la tumeur par les lymphocytes T CD8+. Nous avons étudié la possibilité que l'hyperploïdisation puisse également affecter l'antigénicité des cellules cancéreuses en modifiant l'immunopeptidome. Cependant, la vaccination avec des peptides candidats n’a pas réussi à induire des réponses inhibitrices de la croissance tumorale de cellules hyperploïdes in vivo. Nous concluons donc que l'hyperploïdie augmente principalement l'immunogénicité des cellules cancéreuses en affectant leur adjuvanticité (via l’expression de calréticuline) plutôt que leur antigénicité. Nous montrons de plus que des fibrosarcomes induits par méthylcholanthrène (MCA), développés dans des souris Rag2-/-γc-/- immunodéficientes (mais pas dans des souris sauvages), sont particulièrement hyperploïdes, en corrélation avec une teneur en ADN plus élevée, une augmentation de la surface nucléaire, ainsi que l'hyperphosphorylation de eIF2α,. De telles cellules forment des tumeurs dans les souris Rag2-/-γc-/- (dépourvues de cellules T, B et NK) ainsi que dans les souris Rag2-/- (dépourvues de lymphocytes T et B) sans différence entre les deux souches de souris, suggérant que l'absence de cellules B et T est suffisante pour que ces cellules prolifèrent. Pour mesurer ces paramètres, nous avons développé un outil d'analyse morphométrique applicable à l'immunohistochimie. Cet algorithme identifie et quantifie automatiquement la surface des noyaux et l'intensité de la phosphorylation de eIF2α dans une région d'intérêt périnucléaire. Des analyses comparatives ont validé la précision de cette méthode, qui peut être utilisée pour étudier la ploïdie et le stress du RE dans les cancers in situ.

Introduction: Genetic characterization of malignant mesothelioma shows a homozygous deletion of the INK4A/ARF locus, which results in inactivation of the p53 pathways. Methods: We examined possible antitumor effects of adenoviruses with a deletion of the E1B-55kD gene (Ad-delE1B55) on mesothelioma and investigated combinatory actions with the first-line chemotherapeutic agents. Results: Ad-delE1B55 produced cytotoxicity on mesothelioma cells, which was associated with p53 phosphorylation, pRb dephosphorylation, and cleavage of caspases. Ad-delE1B55–infected cells displayed hyperploidy at the cell-cycle analysis and showed enlarged nuclear configurations. Combination of Ad-delE1B55 plus cisplatin or pemetrexed produced antitumor effects in vitro. Furthermore, Ad-delE1B55 and cisplatin showed combinatory effects in an orthotopic animal model. Conclusions: Cell death caused by Ad-delE1B55 is attributable to cell-cycle arrest at M-phase checkpoint followed by activated apoptotic pathways, and combination of the first-line chemotherapeutic agents and the oncolytic adenovirus is a potential therapeutic for mesothelioma.

Urothelial carcinomas arising from the upper urinary tract (renal pelvis and ureter) are rare and few molecular genetic studies of these tumors have been conducted to date. We investigated hyperploidy at chromosomes 3, 7, and 17 using a multitarget fluorescence in situ hybridization system to identify genetic alterations in patients with urothelial carcinomas of the upper urinary tract. Chromosomal aberrations are seen most frequently in the high-grade tumors. A highly significant relationship was found between an increase in the percentage of hyperdiploidy and high grade for each chromosome (chromosome 3, P = 6 × 10(-4); chromosome 7, P = 2 × 10(-4); chromosome 17, P = 6 × 10(-5)). To determine whether these associations were independent for each chromosome, the correlation between percentage of hyperdiploidy for each pair of chromosomes was examined. In each case, the correlation was highly significant (R = 0.89-0.91). No statistically significant association was found between percentage of hyperdiploidy and tumor stage for any chromosome.

A group of six plants of Allium cepa L., a diploid species with 2n = 16, growing in Kiharian village of Jammu province was studied for somatic and meiotic details. While the somatic cells of these plants possessed normal chromosome count, 14.75% of their pollen mother cells had 16 plus 1–4 chromosomes. These plants were also peculiar in having 12.49 and 8.78% chromosomes associating as multivalents (trivalents to hexavalents) at diakinesis and at metaphase I respectively. Segregation of chromosomes was irregular at anaphase I and pollen stainability very low. Besides, describing meiotic anomalies in detail, the mechanisms likely to be responsible for the same are discussed in the present communication.

The present study was undertaken to analyze the impact of epigenetic alterations with a main focus on nuclear area, aneuploidy, hyperploidy, and proliferation in 70 ovarian cancer specimens.Morphometric changes and somatic chromosomal ploidy status were assessed by Feulgen spectrophotometry. DNA-hypomethylation of LINE1 repeats was analyzed by means of MethyLight PCR, and methylation levels of satellite 2 (Sat2) and satellite alpha (Satα) DNA sequences in chromosome 1 were measured by Southern blot analysis. These parameters were analyzed with regard to correlations as well as to recurrence and survival.We identified a significant association between LINE1 DNA-hypomethylation and patient age (p=0.029). Furthermore, LINE1 DNA-hypomethylation was positively correlated with the nuclear area (r=0.47; p0.001) and the proliferation index (r=0.36; p0.001). Univariate survival analysis showed that the nuclear area and LINE1 DNA-hypomethylation were prognostic factors for overall (p=0.015 and =0.006, respectively) and progression-free survival (p=0.020 and p=0.001 respectively), the percentage of aneuploidy only for overall survival (p=0.031). Subgroup survival analyses revealed that the prognostic value of these factors is strictly confined to mucinous cancers. In serous cancers no prognostic value could be pointed out for any analyzed parameter. Multivariate analysis of the entire cohort showed that the percentage of hyperploidy was an independent prognostic parameter for overall survival (p=0.003) and LINE1 DNA-hypomethylation for progression-free survival (p=0.03). In mucinous cancers nuclear area and LINE1 DNA-hypomethylation were found to be independent predictors of progression-free and overall survival.In this study we identified the correlations between early cancer-associated genome DNA-hypomethylation, nuclear morphometric changes, somatic chromosomal ploidy status and the proliferation index. Prognostic relevance of nuclear area and LINE1 DNA-hypomethylation was revealed exclusively in mucinous ovarian cancers.

Aneuploidy is commonly observed in breast cancer and is associated with poor prognosis. One frequent type of aneuploidy, hypertetraploidy, may derive from ploidy duplication of hyperdiploid cells. However, the pathological consequences of ploidy duplication in breast cancer progression have not been characterized. Here, we present an experimental system demonstrating spontaneous appearance of hypertetraploid cells from organ-specific metastatic variants of the MDA-MB-231 breast cancer cell line through ploidy duplication in vitro and in vivo. The hypertetraploid progenies showed increased metastatic potential to lung and brain, but not to bone, which may be partially explained by the distinct capillary structures in these organs that confer differential lodging advantages to tumor cells with enlarged size. Our results suggest a potential mechanistic link between ploidy duplication and enhancement of metastatic potentials, as was observed in previous clinical studies of breast cancer.

Aneuploidy, an abnormal number of copies of a genomic region, might be a significant source for neuronal complexity, intercellular diversity, and evolution. Genomic instability associated with aneuploidy, however, can also lead to developmental abnormalities and decreased cellular fitness. Here we show that neurons with a more-than-diploid content of DNA are increased in preclinical stages of Alzheimer's disease (AD) and are selectively affected by cell death during progression of the disease. Present findings show that neuronal hyperploidy in AD is associated with a decreased viability. Hyperploidy of neurons thus represents a direct molecular signature of cells prone to death in AD and indicates that a failure of neuronal differentiation is a critical pathogenetic event in AD.

Recently it has been suggested that the human brain contains aneuploid cells; however the nature and magnitude of neural aneuploidy in health and disease remain obscure. Here, we have monitored aneuploidy in the cerebral cortex of the normal, Alzheimer's disease (AD) and ataxia telangiectasia (AT) brain by molecular cytogenetic approaches scoring more than 480,000 neural cells. Using arbitrarily selected set of DNA probes for chromosomes 1, 7, 11, 13, 14, 17, 18, 21, X and Y we have determined the mean rate of stochastic aneuploidy per chromosome as 0.5% in the normal human brain (95%CI 0.2–0.7%; SD 0.2%). The overall proportion of aneuploid cells in the normal brain has been estimated at approximately 10%. In the AT brain, we observed a 2-to-5 fold increase of stochastic aneuploidy randomly affecting different chromosomes (mean 2.1%; 95%CI — 1.5–2.6%; SD 0.8%). The overall proportion of aneuploid cells in the brain of AT individuals was estimated at approximately 20–50%. Compared with sex- and age-matched controls, the level of stochastic aneuploidy in the AD brain was not significantly increased. However, a dramatic 10-fold increase of chromosome 21-specific aneuploidy (both hypoploidy and hyperploidy) was detected in the AD cerebral cortex (6–15% versus 0.8–1.8% in control). We conclude that somatic mosaic aneuploidy differentially contributes to intercellular genomic variation in the normal, AD and AT brain. Neural aneuploidy leading to altered cellular physiology may significantly contribute to the pathogenesis of neurodegenerative diseases. These data indicate neural aneuploidy to be a newly identified feature of neurodegenerative diseases, similar to other devastative disorders hallmarked by aneuploidy such as chromosome syndromes and cancer.

To clarify the characteristics of late-arising (delayed) chromosome aberrations after irradiation in human lymphocytes, 30 B-cell lines were established from the peripheral blood from a healthy adult donor, the lymphocytes of which were exposed to alpha-rays or gamma-rays and then used for experiments. Chromosome aberrations were serially observed at several passages by both conventional cytogenetics and fluorescence in situ hybridization analysis using subtelomere probes. These B-cell lines derived from lymphocytes with a history of radiation exposure had higher percentages of delayed chromosome aberrations, such as dicentrics, rings, endoreduplication, hyperdiploid, hyperploidy, and telomere association. Furthermore, alpha-ray exposure induced higher chromosome instability than gamma-ray exposure, indicating that delayed chromosome aberrations were related with radiation quality. Chromosome instabilities were also observed at the subtelomere. Cell lines showing high chromosome instability had high DNA-PK activity, low expressions of Ku70, p53, and TRF1 proteins after stimulation with radiation. These results indicate that mechanisms underlying delayed chromosome aberrations might be epigenetic, and multiple factors such as defects of DNA-PK, subtelomere, and telomere might be associated.

A cell line designated HTMMT was established from the human uterine carcinosarcoma (composed of leiomyosarcoma and adenocarcinoma) of a 66-year-old Japanese woman. The cell line grew well and 83 serial passages were successively done within 24 months. The cell line contained spindle- or fibrous-shaped cells that revealed neoplastic and pleomorphic features, and multipled rapidly without contact inhibition. These cells were characterized as possessing myofibrils. The karyotype exhibited hyperploidy and the chromosome number was ranged from 87 to 100. The cells were transplanted into an immune-depressed hamster cheek pouch or into nude mouse skin, but produced no tumors. We supported the combination theory for the histogenesis of the carcinosarcoma.

The aneuploidy frequencies have been stuided in vitro in lymphocytes from couples with recurent abortions, from parents of a trisomic child and from control couples with at least two normal children; c-heterochromatin variants have been analysed on the same samples by length measurements. A significant increase of hyeperploid cells has been observed in the lymphocytes from parents of a trisomic and couples with recurrent abortions as well. However, no consistent correlation has been found so far between c-heterochromatin varients and an increase of aneuploidy.

An increase in the incidence of aneuploidy is well documented with increasing maternal age, in particular in human females. Remarkably, little is known regarding the underlying molecular basis for the age-associated increase in aneuploidy, which is a major source of decreased fertility in humans. Using mouse as a model system we find that eggs obtained from old mice (60-70 weeks of age) display a 6-fold increase in the incidence of hyperploidy as assessed by chromosome spreads. Expression profiling of transcripts in oocytes and eggs obtained from young and old mice reveals that approximately 5% of the transcripts are differentially expressed in oocytes obtained from old females when compared to oocytes obtained from young females (6-12 weeks of age) and that this fraction increases to approximately 33% in eggs. The latter finding indicates that the normal pattern of degradation of maternal mRNAs that occurs during oocyte maturation is dramatically altered in eggs obtained from old mice and could therefore be a contributing source to the decline in fertility. Analysis of the differentially expressed transcripts also indicated that the strength of the spindle assembly checkpoint is weakened and that higher errors of microtubule-kinetochore interactions constitute part of molecular basis for the age-associated increase in aneuploidy in females. Last, BRCA1 expression is reduced in oocytes obtained from old females and RNAi-mediated reduction of BRCA1 in oocytes obtained from young females results in perturbing spindle formation and chromosome congression following maturation.

Objective: to study the impact of chromium levels on cytogenetic impairments in T lymphocytes in miners from the south of Kuzbass. Subjects and methods. Peripheral blood leukocytes were cultured, as described by P. S. Moorhead (1960), by using the cytogenetic assay of the obtained specimens. Analysis of chromosomes considered changes in the number of chromosomes (hyper-, hypo-, or polyploid sets of chromosomes) and their structural derangement. Ninety-three miners from Novokuznetsk and Mezhdurechensk (Kemerovo Region) were examined. According to the length of underground service, the miners were divided into 2 groups: 1) 32 miners with a service length of 1—5 years; 2) 31 with a service length of 10—15 years. A control group included 30 employees from the mine maintenance departments. The mean age of the miners was 38.6±3.4 years. Previous coal chemical analysis in a number of mines revealed significant concentrations of chromium compounds (as high as 298 mg/kg in the clarke of 83 mg/kg). Lymphocytic chromium was quantified by the atomic emission method using inductively bound plasma. The statistically data were processed using a package of Statistica 5.01 computer programs. Results. Cytogenetic study and measurement of peripheral blood chromium were made in miners with a various underground service length. They were found to have elevated peripheral blood T lymphocytes with cytogenetic disorders, which correlated with the increased blood chromium levels. Conclusion. Thus, the miners with an underground service length of 10—15 years were ascertained to have high blood chromium levels, which was accompanied by increases in the count of both cells with chromosomal structural aberrations and those with poly- and hyperploidy.

Brain tumor entities are characterized by specific copy number alterations (CNA), allelic losses/disbalances and recurrend mutations. Emerging technologies including SNP arrays, whole exome and whole genome sequencing are not suitable for degraded DNA derived from formalin-fixed, paraffin embedded (FFPE) samples. The aim of our study was to analyse the sensitivity and robustness of molecular inversion profiling (MIP) as a tool to identify these alterations in brain tumors and to compare this method to FISH and multiplex ligation probe analysis (MLPA). Genomic DNA extracted from up to 20 years old FFPE materials from more than 1300 brain tumors covering most WHO entities were analyzed by MIP profiling (Oncoscan V2/V3, Affymetrix). MIP revealed genome-wide copy number information from as little as 20 ng of degraded DNA; drop-out rate was

The sphingolipid ceramide has been recognized as an important mediator in the apoptotic machinery, and its efficient conversion to glucosylceramide has been associated with multidrug resistance. Therefore, inhibitors of glucosylceramide synthase are explored as tools for treatment of cancer. In this study, we used d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol to sensitize Neuro-2a murine neuroblastoma cells to the microtubule-stabilizing agent paclitaxel. This treatment resulted in a synergistic inhibition of viable cell number increase, which was based on a novel mechanism: (a) After a transient mitotic arrest, cells proceeded through an aberrant cell cycle resulting in hyperploidy. Apoptosis also occurred but to a very limited extent. (b) Hyperploidy was not abrogated by blocking de novo sphingolipid biosynthesis using ISP-1, ruling out involvement of ceramide as a mediator. (c) Cyclin-dependent kinase 1 and 2 activities were synergistically decreased on treatment. In conclusion, instead of inducing apoptosis through ceramide accumulation, d,l-threo-1-phenyl-2-decanoylamino-3-morpholino-1-propanol by itself affects cell cycle–related proteins in paclitaxel-arrested Neuro-2a cells resulting in aberrant cell cycle progression leading to hyperploidy. [Mol Cancer Ther 2006;5(3):593–601]

Healthy mice were orally fed with different doses of 3 types of pesticides namely, Metacid (organophosphate insecticide), Profenofos (organophosphate insecticide) and Nimbecidine (biopesticide). The doses were continuously given for consecutive 15 days. Control mice were fed with water. All the doses were given at the rate of 1 ml/100 g body weight. The genotoxic effects in pesticide-treated mice were assessed through the study of chromosomal aberrations, sperm head anomaly as against suitable untreated controls. The observations revealed that chromosomal abnormalities viz, chromosome fragment, somatic reduction, hypoploidy, hyperploidy etc. as well as abnormal sperm heads were found in pesticide-fed mice and maximum abnormalities were associated with Profenofos.

The chromosome set of human spermatozoa was studied by intracytoplasmic injection into mouse oocytes. A total of 85 metaphase plates of male pronuclei of a patient with chromosome constitution 45,X/46,X,r(Y) and 108 metaphase plates of patients with normal sperm parameters (control group) were examined. The ratio between X- and Y-bearing chromosomes in the 45,X/46,X,r(Y) patient and in the control group did not differ from 1 : 1. A significant increase in the rates of diploidy, hypoploidy, hyperploidy of sex chromosomes, and chromosome structure rearrangements in spermatozoa of the patient in comparison with spermatozoa in the control group was recorded.

Trisomies due to nondisjunction in oogenesis are still a major cause of genetic diseases in humans. In this study, we analysed spindle morphology of in vitro matured nocodazole-exposed mouse oocytes by novel non-invasive Polscope-microscopy, and compared images to those obtained by anti-tubulin immunofluorescence of fixed oocytes. Polscope revealed a reduction in the numbers of oocytes expressing a birefringent spindle, and alterations in spindle morphology at concentrations of nocodazole below those inducing detectable aberrations in immunofluorescence. Hyperploidy increased significantly at a concentration of 40 nM nocodazole in mouse metaphase 11 oocytes, similar to thresholds inducing nondisjunction in cultured human lymphocytes. In conclusion, Polscope represents a novel highly sensitive, non-invasive method to identify chemicals inducing severe spindle aberrations that predispose mammalian oocytes to nondisjunction. Polscope may provide information on the functionality of the spindle in experimental studies but is also compatible with clinical trials in human assisted reproduction due to its non-invasive nature. (c) 2004 Elsevier Inc. All rights reserved.

The chromosome complement of human spermatozoa has been analyzed after their intracytoplasmic injection into unfertilized mouse oocytes. A total of 427 metaphase plates have been obtained, including 176 metaphase plates from spermatozoa with normal head morphology (108 and 68 spermatozoa from patients with normal (the control group) and abnormal spermogram parameters, respectively), and 251 metaphase plates from spermatozoa with abnormal heads (76, 91, 67, and 17 spermatozoa with large, amorphous, elongated, and round heads, respectively). The frequency of chromosome abnormalities in the control group is 26.1%, with hyperploidy, hypoploidy, and structural aberrations accounting for 7.4, 12.3, and 6.4% of the abnormalities, respectively. In none of the groups did the ratio between the numbers of X- and Y-bearing spermatozoa significantly differ from 1 : 1. The diploidy frequency was significantly higher in spermatozoa with large and amorphous heads compared to the control group (2.36, 3.29, and 0%, respectively). None of the groups of spermatozoa differed from the control group with respect to the frequency of structural aberrations. The type of the abnormal head morphology has been found to be correlated with the sperm chromosome complement.

Chromosomal anomalies were assessed in nuclear transfer (NT) embryos (n = 148) at 1-4-cell stage (n = 88), and morula (n = 60), as well as in donor cells (n = 97) derived from two different cell lines. Two different cytogenetic approaches were used: conventional karyotyping and fluorescent in situ hybridization (FISH) with painting probes, specific for bovine X and Y chromosomes. The total rate of NT embryos with abnormal nuclei was 43%. These anomalies were mainly nuclear fragmentation (30%), hypoploidy/hypoploidy-mixoploidy (9%, n = 14) and hyperploidy/hyperploidy-mixoploidy (3%, n = 5). The incidence at which these anomalies occurred in NT embryos varied according to the donor cell culture and paralleled the frequency of anomalies in donor cells. A higher frequency of total anomalies was observed in NT embryos (55%) derived from the donor cell cultures with the highest incidence of anomalies (23%). An increase in the rate of total anomalies of the cell, after transfer to recipient cytoplasm, was also observed. These results suggest that proper screening of donor cells for chromosomal anomalies must be performed prior to NT procedure. They also suggest that the NT procedure itself might have a detrimental effect on some mechanism of chromosome segregation and distribution during cell division.

Much debate exists on factors predicting the development of persistent gestational trophoblastic disease (pGTD). Diagnosis is still limited by following persistently elevated or rising postevacutation beta-human chorionic gonadotropin (beta-hCG) titers. The aim of the present work was to evaluate the hypothesis that the presence of c-erbB-2 oncogene amplification and expression, in combination with parameters such as DNA-content and karyotype of the sex chromosomes, confer an increased risk of developing pGTD. Clinicopathological characteristics were evaluated in 36 cases of gestational trophoblastic diseases (GTD) and analyzed for c-erbB-2 amplification and protein p185 expression using differential polymerase chain reaction (DPCR) and immunohistochemical (IHC) techniques. The DNA-content was determined by image analysis on Feulgen stained nuclear cell preparations and karyotyping for XY chromosomes was performed by fluorescence in situ hybridization (FISH). The data was correlated with histopathological characteristics of GTD. Seventy-five percent (n = 27) of the examined cases showed spontaneous regression after evacuation, including 2 patients who received additional chemotherapy. Twenty-five percent (n = 9) resulted in a persistent or metastatic disease. The median time between antecedent pregnancy and GTD was 45.4 months. Complete remission was achieved in all patients with pGTD after administration of chemotherapeutic agents or adjuvant surgical procedures. Cases with cerbB-2 amplification and expression in combination with DNA hyperploidy showed higher proliferation and more aggressive behavior (2 complete hydatidiform moles with lung and liver metastases, 2 invasive moles and 1 choriocarcinoma). XY karyotype was evident in the choriocarcinoma and in 2 complete hydatidiform moles with advanced stage and DNA hyperploidy. From these results we conclude that c-erbB-2 amplification and/or protein expression in combination with DNA-content show a significant correlation with the proliferative and aggressive potential of GTD, suggesting their combined use as a possible marker for pGTD.