Analysis Of Metaphase Chromosomes Research Articles

Study of the Effect of Plavix on Micronuclei in Albino Mice

The advanced basic science of toxicogenomics is a branch of toxicogenetics, aimed at self-engaging acoustic factor activation of advanced components of customized systems. The PCES micronuclei test provides a rapid and accurate way to detect the genotoxicity of chemical compounds that cause a clastogenic or anogenic effect, and it is also of statistically significant value because it can register a much larger number of cells compared to the metaphase chromosome analysis test. Except that he is The other cannot detect microscopic lesions in the cells' DNA. Adopting the technique of estimating micronuclei in exfoliated epithelial cells in prior monitoring of the occurrence of tumors Cancer and treatment response monitoring programmes. 2 Adopting micronuclei technology in expanded programs to screen chronic disease groups for toxicological agents Genetic.

Study of antimutagenic activity of medicinal plant infusions Crataegus sanguinea Pall. family Rosaceae in plant test systems

Medicinal plants, integral to traditional medicine systems, are rich sources of biologically active substances that benefit physiological and biochemical processes within living organisms. Amongst these activities, antimutagenic and genoprotective properties stand out, offering mitigation against genotoxic effects induced by adverse environmental factors on genetic material. This study delves into the mutagenic and antimutagenic capacities of aqueous and alcoholic infusions derived from Crataegus sanguinea Pall. (Rosaceae family), utilising Hordeum vulgare L. as a plant-based test subject. The assessment employed the metaphase chromosome analysis technique. Results indicate that these infusions exhibit no mutagenic activity, with the level of chromosomal aberrations in barley seeds treated with these infusions not exceeding the natural mutation rate in a statistically significant manner. When infusions were combined with Methyl methanesulfonate (positive control) exposure – irrespective of exposure sequence – a statistically significant attenuation in MMS-induced mutagenesis was observed (p<0.01). A 56-60% reduction quantified the antimutagenic efficacy of C.sanguinea infusions. This metric underscores the infusions’ capability to inhibit MMS-induced mutagenesis by 50–60%, positioning these water and alcohol-based extracts of common hawthorn as viable candidates for safeguarding against chemically induced mutagenic factors.

Polyploidy in the history of breeding at the Yaltushkiv Experimental Breeding Station: new methods of differentiation and stabilisation of sugar beet tetraploids

Purpose. To analyse the history of the polyploid breeding at the Yaltushkiv Experimental Breeding Station, in particular the improvement of genetic models of new hybrids, new methods of induction of meiotic tetraploids, differentiation and selection of breeding genotypes by genome ploidy.
 Methods. Cytological analysis of metaphase chromosomes of the apical meristems of the growth points of seed plants and beets of the first year for the selection of tetraploid plants 4x = 36 chromosomes; cytophotometric analysis using computer programs AP Partec to study ploidy based on the quantitative content of nuclear DNA; field methods (selection of tetraploid seed plants according to the types of pollen grains, formation of synthetic populations of tetraploid multigerm plants on a cytogenetic basis according to the structure of bivalent conjugation of homologous chromosomes in meiosis.
 Results. In the process of tetraploid induction, cytological methods of ploidy analysis at the station were improved using computer programs of AP Partec for the selection of sugar beets 4x by the quantitative content of nuclear DNA in the cell on plants of the first year of vegetation and for the formation of tetraploid populations after colchicine induction in seed plants. In the process of studying the structure of polyploid populations by ploidy, the percentage of tetraploids varied from 82.0±2.22% in Ya/Hen pollinator to 58.1±2.81% in Ya/Man pollinator. It was established that the selection for fertility and pollen uniformity depends on the genetic characteristics of the breeding genotypes and affects the pollen-forming ability in the first generations after induction with colchicine, reducing the number of 4x IIa and IIb biotypes according to cytological classification. Using the example of the multigerm pollinator Ya/Roi, it was established that selection for sugar content and productivity had, to a certain extent, a stabilising effect on the degree of ploidy. The biotechnological methods used to deposit 4x genotypes with a high percentage of bivalent conjugation of homologous chromosomes in metaphase I of meiosis (70–90%) contributed to the cytogenetic stability of polyploid gametes and their high yield – 92.9% of 4x in the next generation of seeds. It was investigated that the productivity of triploid hybrids obtained with the use of the tetraploid pollinator Ya/DA 4x based on the germplasm of the wild species Beta patula at the early stages of vegetative development varied in triploid plants from 325 to 465 g, and the sugar content in some numbers reached 21.5%.
 Conclusions. The development of polyploid selection at the Yaltushkiv Experimental Breeding Station evolved from the development polyhybrids to the development of triploid hybrids based on CMS using tetraploid multigerm pollinators. For the formation of populations of tetraploid multigerm pollinators, the methods of fluorescent cytophotometry and computer programs AP Partec were used, as well as biotechnological methods for creating synthetic populations of tetraploid multigerm pollinators on a cytogenetic basis, which ensure the yield of tetraploids in seeds up to 92.9%.

Genome Stability Profiles of Titania Nanosurface Technology for Biomedical Applications

The emergence of nano-based products for advanced biomedical applications has underlined the need for toxicogenomic studies which govern genome stability endpoints. In the present study, the genome stability profiles on cell-TNA interaction were assessed via nuclear staining profile by a fluorescent microscope, DNA ploidy profile by flow-cytometry, metaphase chromosome analysis, DNA topoisomerase II alpha (TOP2A) profiling by chromogenic in situ hybridization (CISH), cytokinesis-blocked micronucleus assay (CBMN), reactive oxygen species (ROS) measurement and gene expression profile by real-time polymerase chain reaction (RT-PCR). Findings from this study demonstrated that no significant genomic instability risk was observed from cell-TNA interaction, especially at clastogenic and aneugenic levels. Findings also suggest that cell-TNA interaction could involve the regulation of ribonucleoprotein complex and cell metabolism associated with the cellular adaptation process towards the nanosurface. Further comprehensive studies involving in vivo models are needed to support this work.

Analysis of the Chromosomal Aberrations in Hepatitis B Virus (HBV), Hepatitis C Virus (HCV), and Hepatocellular Carcinoma Patients (HCC) in Mosul City, Iraq

Abstract. Hepatocellular carcinoma (HCC) (also known as liver cancer) is one of the most frequent cancers in humans. HCC is linked to chronic hepatitis B and C virus infection, cirrhosis, and excessive alcohol consumption. The aim of this study was to use Metaphase chromosome analysis in whole blood to determine chromosomal aberrations (CA) in HBV, HCV, and HCC patients. A cohort of 145 samples have been collected from participants from the date of 5 \ 1 \ 2020 to 15\9\ 2021. Among those samples are (40 healthy controls, HBV 38, 44 HCV, and HCC 23) to make cytogenetic evaluation by observing the analysis of chromosome aberration. Our study showed that the chromosome aberration With Gap was higher in the HCC patient group followed by HBV patient group. also, the results showed that of the chromosome aberration without Gap in the HCC, and HCV patient groups were significantly higher in females than in males. the results showed that higher break, DIC was in HBV patients group followed by HCV patients group. the results showed that deletion, and ace were higher in the HCV and HCC patient group followed by HBV patient group. Finally with RO. Trans., the results showed that ace was higher in the HCV, and HBV patient groups. In conclusion, we indicate that HBV, HCV, and HCC patients have chromosomal instability because of their chromosome aberration levels.

Cluster analysis of karyotype similarity coefficients in Epimedium (Berberidaceae): insights in the systematics and evolution.

In order to evaluate the genome evolution and systematics, karyotype analysis of mitotic metaphase chromosomes in 51 taxa of Epimedium and two species of Vancouveria was conducted. The 53 taxa were clustered, based on their karyotype similarity coefficients. Results showed that the 53 taxa studied were all diploid with 12 chromosomes (2n = 2x = 12). Each taxon had one pair of satellites located on pair I of homologous chromosomes. Moreover, the karyotype types of the 53 taxa studied were all type 1A or 2A of Stebbins. It can be concluded that the karyotypes between species are indeed very similar and the genome of Epimedium was conservative in evolution. The cluster analysis of karyotype similarity coefficients could provide valuable clues for the systematics and taxonomy of Epimedium. Results of the cluster analysis strongly supported the previous taxonomic division of E. subg. Rhizophyllum and E. subg. Epimedium. The results also showed that the interspecific relationship was closely correlated with geographical distribution in E. subg. Epimedium and the taxa native to east Asia had the highest genetic diversity in Epimedium. Finally, the origin of the modern geographical distribution of Epimedium was inferred. Results of the present study have significant scientific values in further studies on resource utilisation, taxonomy and phylogeny in Epimedium.

Rapid fluorescence in situ hybridisation optimises induction therapy for acute myeloid leukaemia.

Rapid fluorescence in situ hybridisation optimises induction therapy for acute myeloid leukaemia.

Whole genome SNP arrays for best practice for detection of diagnostic, prognostic and therapy related copy number changes and copy neutral-loss of heterozygosity across solid tumors and hematologic malignancies.

e15575 Background: High resolution single nucleotide polymorphism (SNP) chromosomal microarrays (CMA) detect copy number changes and copy neutral-loss of heterozygosity (CN-LOH) across the entire genome, currently providing the best assessment for these types of genomic variants. Chromosomal microarrays are first tier tests utilized in the postnatal detection of microdeletions, microduplications and uniparental disomy/regions of homozygosity in constitutional disorders involving congenital abnormalities, developmental delay and intellectual disability. Methods: In the oncology setting, aberrations detected may be diagnostic, prognostic, and therapeutic. Because CMA assesses the entire genome and can readily detect aberrations as small as a single exon to as large as a whole chromosome, this is an important clinical tool to bridge the gap between low resolution of metaphase chromosome analysis and PCR-based short read sequencing-based assays. Results: No single genomic technique (metaphase chromosome analysis, FISH, CMA or Next Generation Sequencing, including large targeted gene panels) has the ability to detect all relevant information. Therefore, CMA should be considered an important clinical tool for solid and liquid tumors. Across a wide variety of solid tumors, whole genome assessment (including oncogene amplification, tumor suppressor loss, and copy number burden) leads not only to possible therapy targets but also to opportunities for participation in active clinical trials. Recently, the Cancer Genomics Consortium has published evidenced-based reviews on the clinical utility of CMA for copy number and CN-LOH assessment in a variety of hematologic malignancies, and similar papers in solid tumors are in review. Recognizing the growing evidence for CMA, the American Medical Association (AMA) CPT editorial board recently created a new Tier 1 test for cytogenomic arrays in neoplasia, and Centers for Medicare and Medicaid Services (CMS) approved crosswalking the price of the new code to the well-established constitutional cytogenomic array CPT code. Conclusions: For this presentation, examples of diagnostic, prognostic, and therapeutic utility and inclusion in clinical trials across many hematologic and solid tumor neoplasms will be presented to demonstrate the efficacy, cost effectiveness and sensitivity of whole genome assessment of copy number and copy neutral loss of heterozygosity

Frequency of Chromosomal Aberrations in Somatic Cells of Ukrainian Buffaloes (Bubalus bubalis L.)

Routine, GTG-, and Ag-staining techniques for the metaphase chromosome analysis were used to determine the spontaneous frequency of chromosomal aberrations and the level of chromosomal variability in the blood lymphocytes of Ukrainian buffaloes (Bubalus bubalis L.). It has been established that the diploid complement of chromosomes within the studied population of animals consists of 50 chromosomes (2n = 50, XX; 2n = 50, XY). Individual chromosomal variability was detected in some animals in the form of cells with aneuploidy and polyploidy sets of chromosomes as well as in the form of structural aberrations in autosomes. In particular, the chromosomal analysis based on differential staining technique has shown a duplicate in one chromosome of the second pair in individual females as well as an X monosomia and a chimerism over sex chromosomes. The total spontaneous level of chromosomal instability in the peripheral blood lymphocytes reached, on average, 14.35 ± 2.03% due to abnormalities of chromosomal and chromatid types. The Ag staining method was used to detect active NORs in six chromosome pairs (3p, 4p, 6q, 21q, 23q, and 24q).

GENDER PECULIARITIES OF THE SPECTRUM OF CHROMOSOMAL ABERRATIONS IN PERIPHERAL BLOOD LYMPHOCYTES OF HEALTHY PERSONS AND PATIENTS WITH GENERALIZED PERIODONTITIS

Introduction. Generalized periodontitis (GP) refers to the diseases with multifactorial heredity, in which the variability of one or another sign is determined not by one major gene, but by the influence of a large number of hereditary and external factors with small effects. An important component for the research of immuno-cytogenetic status of healthy persons and patients with multifactorial diseases is the study of chromosomal abnormalities. For this purpose, a method of metaphase analysis of karyotype in peripheral blood lymphocytes is used.
 Aim is to investigate gender peculiarities of the frequency and spectrum of chromosomal aberrations (CA) in peripheral blood lymphocytes of GP patients with chronic course of various degrees of development.
 Methods. There were examined 54 patients, who were divided into three groups: 18 persons (8 men and 10 women) healthy (group I); 24 persons (by 12 men and women) – patients with the GP of the initial-I degree (group II) and 12 persons (by 6 men and women) with II-III degree of GP (group III).
 Analysis of metaphase chromosomes was performed in venous blood according to the methodology of the Ministry of Health of Ukraine in the accredited genetic laboratory of the State Medical University, SHEI “Ivano-Frankivsk National Medical University” after lymphocytes cultivation in the nutrient medium «PB-max» during 72 hours at a temperature of +37ºС. In each preparation, at least 100 metaphases with a good chromosomal dispersion were analyzed. Microscopic examination was performed using a microscope «Axioskop» company Zeizz (magnification×1000) at the optical-electronic complex “Metacan-2”. The frequency and spectrum of CA in men and women in terms of gaps, ruptures, single and paired fragments, translocations, deletions, dicentricks were studied.
 Results. It has been determined, that the frequency of CA increased with the degree of GP development (p<0.001; p1<0.001) and did not significantly depend on the gender, however, there was a tendency to increase the number of CA in women of all groups. In this case, the presence of gender dimorphism in the CA spectrum in GP patients was found with the increase of the frequency of gaps in all men, depending on the increase of the disease development degree (p<0.05; p<0.01), while in women with II-III degree the number of such aberrations decreased in relation to the indicators of GP patients of the initial-I degree. We’ve also discovered the characteristic gender peculiarities of chromosomal aberrations – paired fragments. Their number in men with GP of II-III degree was lower than the indices of patients with GP of the initial-I degree, and in women – it was higher. Gender dimorphism was manifested in relation to translocations that were not manifested in healthy men at all, while in the ill women of both groups the indices were prevalent than those in men.
 Some gender difference is also a significant increase in the proportion of gaps (p≤0.001) and deletions among all CA in women suffering from GP of both degrees, whereas in the ill men, the number of this type of aberrations increased slightly. A gender peculiarity has also been determined regarding the important marker of CA of chromatid type – single fragments. The frequency of such aberrations in men with GP gradually increased, depending on the degree of the disease development, while women tended to reduce the number of single fragments in the case of GP of the initial-I degree and their certain increase in GP of II-III degree compared with healthy ones.
 Conclusion. Most of the CA were found in all of the examined patients, however, in GP patients they were significantly more. In this case, in an increase of the degree of the disease development, changes in the structure of the chromosomes occurred more often. The presence of dicentric chromosomes (dicentrics) in patients with GP of II-III degree of men and women showed instability of the genotype, which caused the violation of the implementation of genetic information. An increase in the number of CA in the GP may indicate a slight weakening of the protective forces of the patients’ bodies, because this precludes the elimination of cells with a broken genetic apparatus.

Animal Lymphocyte Metaphase Chromosome Preparation.

Metaphase chromosome analysis of lymphocytes is the gold standard for biodosimetry to estimate the levels of radiation exposure in various animals as well as humans. Animals, including experimental, companion, and wild animals, are powerful and indispensable models for researching radiation injury, safety, and therapy. Moreover, biodosimetry of animal models can be used to support human biodosimetry data and may be useful for estimating environmental contamination by radioactive materials. The basic restraint procedure and venipuncture technique are different depending on each animal type. The general procedure evaluating metaphase chromosomes is similar to the human blood technique except for a minor modification in the initial culture. This chapter will introduce basic mouse, rat, rabbit, dog, cat, cow, horse, goat, pig, and wild boar venipuncture and blood sampling techniques for metaphase chromosome preparation and analysis.

Overlapping Chromosome Segmentation using U-Net: Convolutional Networks with Test Time Augmentation

An effective human metaphase chromosome analysis system can be used by doctors as a second opinion during diagnosis. Segmentation is necessary in developing this system to identify and distinguish between individual chromosomes. The main challenge in chromosome segmentation is the separation of overlapping chromosomes. Deep convolutional neural networks have been widely used for medical segmentation, especially with U-Net. This study investigated how Test time augmentation with a suitable number of U-Net layers can improve the design for this semantic segmentation problem. The proposed architecture was trained, validated and tested with 13,434 greyscale images with 88 × 88 pixels of overlapping chromosome pairs. With the implementation of the proposed method, the training result became more accurate without any mislabelling and additional pre-processing became unnecessary. An improved segmentation accuracy of 99.68% was obtained, which was higher than the 99.22% obtained using the method of Hu et al.

Kanserli hücre hatları, pasaj sayısı arttıkça genomik organizasyonunu ve karyotipini değiştirir: sitogenetik bir çalışma

Purpose: The limited use of mammals in human health related scientific research has led to the development of new research strategies like cell culture techniques. Commercially available cancerous cell lines that are well characterized by cytogenetics and biochemical markers allow comparison of results among different laboratories. However, as these cell lines tend to be maintained in culture over long periods of time, mutations can occur that may change characteristics and responses of cell lines that have initially been identified or non-existed at earlier passages. Here we cytogenetically investigated the chromosomal rearrangements in repeated cultures of six different cell lines over continuous passages. Method: MCF7, HCT116, A549, SHSY5Y, HEPG2, and NIH3T3 cell lines were cultured in DMEM containing 10% FBS and 1% penicillin-streptomycin. GTG banding procedure was used for the analysis of metaphase chromosomes, at least 20 metaphases were analyzed per cell line. Results: We found chromosome number variations and structural changes in the all examined cell cultures as the passage numbers increase. Conclusion: Cell lines have long been used in research to test drugs, to delineate molecular mechanisms, to understand the environmental effects and so on. The most important feature of a cell line is its genotype and karyotype similarities with their host organism. Cancer Cell lines, possess genomic/chromosomal instability that also lead them to change their phenotype along with their karyotype from one passage to next. Therefore, it is always best to verify karyotype before employing a specific cell line in a research project.

Limited FISH Testing for MDS-Defining Cytogenetic Abnormalities Rapidly Identifies Patients with Newly Diagnosed AML Eligible for CPX-351

▪Introduction: CPX-351 (liposomal cytarabine and daunorubicin; VYXEOS) improves survival and complete remission rates compared to standard induction chemotherapy (7+3) in patients 60 to 75 years old with newly diagnosed myelodysplastic syndrome (MDS)-associated and therapy-related AML (Lancet et al., JCO 2018) and was approved by the U.S. Food and Drug Administration in August 2017. In addition to patients with a known history of MDS or cytotoxic and/or radiotherapy, patients with de novo AML with WHO-defined MDS-related cytogenetic changes are also eligible for CPX-351. We have implemented a diagnostic clinical pathway for patients with newly diagnosed AML which includes rapid FISH assessment for the most common MDS-defining karyotypes in patients who are potential candidates for CPX-351. We reviewed outcomes after the first year of this diagnostic pathway in our institutional cohort comparing results of chromosome metaphase analysis and rapid FISH.Methods: We performed both rapid MDS-FISH testing (turnaround time < 6 hours) and metaphase analysis on all patients who presented to the University of Pennsylvania from 9/2017 through 6/2018 with newly diagnosed AML who were potential candidates for CPX-351 induction therapy based on age and other clinical factors. Our MDS-FISH panel includes probes for EGR1(5q31)/5p15.2, D7S486(7q31)/CEP7, and TP53(17p13.1)/NF1(17q11.2) (MetaSystems). Results of the limited FISH testing were compared with metaphase chromosome analysis. Demographic and clinical information was abstracted from the electronic medical record. This study was approved by the University of Pennsylvania Institutional Review Board.Results: Twenty-nine patients with newly diagnosed AML from 9/2017 through 6/2018 had both rapid MDS-FISH and karyotype testing performed. Nineteen patients (65.5%) had de novo AML, while 7 (24.1%) had a clinical history of MDS and 3 (10.3%) had a history of prior cytotoxic and/or radiation therapy. Metaphase analysis revealed WHO-defined MDS-related cytogenetic abnormalities in 14/29 patients (48.3%). Of these 14 patients, 11 (78.6%) also had positive MDS-FISH testing in >=1 probe. Of the 3 patients with MDS-related cytogenetic abnormalities that were identified by metaphase analysis but not the FISH panel, 2 had a history of either MDS (n=1) or prior cytotoxic chemotherapy (n=1) and therefore were eligible for CPX-351 based on clinical history alone. Of the 7 patients with de novo AML and MDS-defining cytogenetic abnormalities, 6 (85.7%) were identified by the FISH screen.Conclusions: Rapid FISH testing for MDS-defining cytogenetic changes using probes for abnormalities of chromosomes 5, 7, and 17 efficiently identifies the majority of patients with newly diagnosed de novo AML who do not have a clinical history of MDS or cytotoxic therapy but are eligible for induction therapy with CPX-351 based on the presence of MDS-defining cytogenetic abnormalities. The combination of rapid FISH testing and clinical history has a high sensitivity for identifying patients with MDS-defining karyotypic abnormalities. This diagnostic clinical pathway allows for the expeditious identification of patients eligible for CPX-351. DisclosuresFrey:Servier Consultancy: Consultancy; Novartis: Consultancy. Perl:Astellas: Consultancy; Daiichi Sankyo: Consultancy; Pfizer: Membership on an entity's Board of Directors or advisory committees; Actinium Pharmaceuticals: Membership on an entity's Board of Directors or advisory committees; AbbVie: Membership on an entity's Board of Directors or advisory committees; NewLink Genetics: Membership on an entity's Board of Directors or advisory committees; Novartis: Membership on an entity's Board of Directors or advisory committees; Arog: Consultancy.

Preparation, Culture, and Analysis of Amniotic Fluid Samples.

Amniotic fluid obtained via amniocentesis provides a source of fetal material used in prenatal diagnosis. The fluid may be used directly for biochemical analyses, fluorescence in situ hybridization (FISH), and isolation of DNA for molecular studies, including chromosomal microarray analysis (CMA). The fluid is typically cultured as a source of metaphase cells for chromosome analysis and to provide additional material for biochemical and DNA-based testing. This unit describes an in situ method for the preparation, culture, and harvest of amniotic fluid samples for metaphase chromosome analysis. Cells are grown, harvested for metaphase spreads, and analyzed on glass coverslips. The unit also describes methods to obtain cells for additional studies (such as molecular genetic analyses) by growing cells in flasks either following passaging of cells from a glass coverslip culture or by directly establishing a flask culture from the amniotic fluid specimen. When cells are grown in flasks, they must be removed from the flask with trypsin before they can be used in studies. Lastly, this unit describes a method for isolating DNA for CMA from uncultured amniotic fluid and cultured cells. © 2018 by John Wiley & Sons, Inc.

Interphase Chromosome Profiling: A Method for Conventional Banded Chromosome Analysis Using Interphase Nuclei.

- Chromosome analysis on bone marrow or peripheral blood samples fails in a small proportion of attempts. A method that is more reliable, with similar or better resolution, would be a welcome addition to the armamentarium of the cytogenetics laboratory. - To develop a method similar to banded metaphase chromosome analysis that relies only on interphase nuclei. - To label multiple targets in an equidistant fashion along the entire length of each chromosome, including landmark subtelomere and centromere regions. Each label so generated by using cloned bacterial artificial chromosome probes is molecularly distinct with unique spectral characteristics, so the number and position of the labels can be tracked to identify chromosome abnormalities. - Interphase chromosome profiling (ICP) demonstrated results similar to conventional chromosome analysis and fluorescence in situ hybridization in 55 previously studied cases and obtained useful ICP chromosome analysis results on another 29 cases in which conventional methods failed. - ICP is a new and powerful method to karyotype peripheral blood and bone marrow aspirate preparations without reliance on metaphase chromosome preparations. It will be of particular value for cases with a failed conventional analysis or when a fast turnaround time is required.

Vemurafenib-resistance via de novo RBM genes mutations and chromosome 5 aberrations is overcome by combined therapy with palbociclib in thyroid carcinoma with BRAFV600E.

PurposePapillary thyroid carcinoma (PTC) is the most frequent endocrine tumor. BRAFV600E represents the PTC hallmark and is targeted with selective inhibitors (e.g. vemurafenib). Although there have been promising results in clinical trials using these inhibitors, most patients develop resistance and progress. Tumor clonal diversity is proposed as one mechanism underlying drug resistance. Here we have investigated mechanisms of primary and secondary resistance to vemurafenib in BRAFWT/V600E–positive PTC patient-derived cells with P16-/- (CDKN2A-/-).Experimental DesignFollowing treatment with vemurafenib, we expanded a sub-population of cells with primary resistance and characterized them genetically and cytogenetically. We have used exome sequencing, metaphase chromosome analysis, FISH and oligonucleotide SNP-microarray assays to assess clonal evolution of vemurafenib-resistant cells. Furthermore, we have validated our findings by networks and pathways analyses using PTC clinical samples.ResultsVemurafenib-resistant cells grow similarly to naïve cells but are refractory to apoptosis upon treatment with vemurafenib, and accumulate in G2-M phase. We find that vemurafenib-resistant cells show amplification of chromosome 5 and de novo mutations in the RBM (RNA-binding motifs) genes family (i.e. RBMX, RBM10). RBMX knockdown in naïve-cells contributes to tetraploidization, including expansion of clones with chromosome 5 aberrations (e.g. isochromosome 5p). RBMX elicits gene regulatory networks with chromosome 5q cancer-associated genes and pathways for G2-M and DNA damage-response checkpoint regulation in BRAFWT/V600E-PTC. Importantly, combined therapy with vemurafenib plus palbociclib (inhibitor of CDK4/6, mimicking P16 functions) synergistically induces stronger apoptosis than single agents in resistant-cells and in anaplastic thyroid tumor cells harboring the heterozygous BRAFWT/V600E mutation.ConclusionsCritically, our findings suggest for the first time that targeting BRAFWT/V600E and CDK4/6 represents a novel therapeutic strategy to treat vemurafenib-resistant or vemurafenib-naïve radioiodine-refractory BRAFWT/V600E-PTC. This combined therapy could prevent selection and expansion of aggressive PTC cell sub-clones with intrinsic resistance, targeting tumor cells either with primary or secondary resistance to BRAFV600E inhibitor.

Novel t(5;11)(q32;q13.4) with NUMA1-PDGFRB fusion in a myeloid neoplasm with eosinophilia with response to imatinib mesylate

We report a NUMA1-PDGFRB fusion in a myeloproliferative neoplasm with eosinophilia in a 61-year old man, with response to imatinib mesylate therapy. A t(5;11) chromosome translocation involving bands 5q32 and 11q13.4 was identified by metaphase chromosome analysis, and rearrangement of the platelet-derived growth factor receptor beta (PDGFRB) gene on 5q32 was demonstrated by FISH using a PDGFRB break-apart probe set. Bacterial artificial chromosome (BAC) FISH mapping of the PDGFRB fusion partner gene narrowed the breakpoint at 11q13.4 to a 150 kb genomic region containing three genes, including NUMA1. Mate pair sequencing analysis demonstrated NUMA1-PDGFRB fusion. The fusion protein includes coiled-coil domains of nuclear mitotic apparatus protein 1 (NuMA1, involved in protein homodimerization and heteroassociation) and tyrosine kinase domains of PDGFRB. Diverse rearrangements involving the PDGFRB gene have been identified in myeloid and lymphoid neoplasms with eosinophilia, but rearrangement of the nuclear mitotic apparatus protein 1 (NUMA1) gene has previously been reported in a human malignancy in only one instance, a NUMA1-RARA fusion caused by a t(11;17) translocation in a patient with acute promyelocytic leukemia. The NUMA1-PDGFRB fusion is the second instance of rearrangement of NUMA1, encoding an element of the mitotic apparatus, in human cancer.

Chromosome sorting for the masses?

Chromosome sorting for the masses?

Repetitive sequence analysis and karyotyping reveal different genome evolution and speciation of diploid and tetraploid Tripsacum dactyloides

In the subtribe Maydeae, Tripsacum and Zea are closely related genera. Tripsacum is a horticultural crop widely used as pasture forage. Previous studies suggested that Tripsacum might play an important role in maize origin and evolution. However, our understanding of the genomics and the evolution of Tripsacum remains limited. In this study, two diploids, T. dactyloides var. meridionale (2n=36, MR) and T. dactyloides (2n=36, DD), and one tetraploid, T. dactyloides (2n=72, DL) were sequenced by low-coverage genome sequencing followed by graph-based cluster analysis. The results showed that 63.23%, 59.20%, and 61.57% of the respective genome of MR, DD, and DL were repetitive DNA sequence. The proportions of different repetitive sequences varied greatly among the three species. Fluorescence in situ hybridization (FISH) analysis of mitotic metaphase chromosomes with satellite repeats as the probes showed that the FISH signal patterns of DL were more similar to that of DD than to that of MR. Comparative analysis of the repeats also showed that DL shared more common repeat families with DD than with MR. Phylogenetic analysis of internal transcribed spacer region sequences further supported the evolutionary relationship among the three species. Repetitive sequences comparison showed that Tripsacum shared more repeat families with Zea than with Coix and Sorghum. Our study sheds new light on the genomics of Tripsacum and differential speciation in the Poaceae family.