Aberrant Metaphases Research Articles

Costunolide induces micronuclei formation, chromosomal aberrations, cytostasis, and mitochondrial-mediated apoptosis in Chinese hamster ovary cells.

Costunolide (CE) is a sesquiterpene lactone well-known for its antihepatotoxic, antiulcer, and anticancer activities. The present study focused on the evaluation of the cytogenetic toxicity and cellular death-inducing potential of CE in CHO cells, an epithelial cell line derived from normal ovary cells of Chinese hamster. The cytotoxic effect denoting MTT assay has shown an IC50 value of 7.56μM CE, where 50% proliferation inhibition occurs. The oxidative stress caused by CE was confirmed based on GSH depletion induced cell death, conspicuously absent in N-acetylcysteine (GSH precursor) pretreated cells. The evaluation of genotoxic effects of CE using cytokinesis block micronucleus assay and chromosomal aberration test has shown prominent induction of binucleated micronucleated cells and aberrant metaphases bearing chromatid and chromosomal breaks, indicating CE's clastogenic and aneugenic potential. The apoptotic death in CE treated cells was confirmed by an increase in the number of cells in subG1 phase, exhibiting chromatin condensation and membranous phosphatidylserine translocation. The apoptosis induction follows mitochondrial mediation, evident from an increase in the BAX/Bcl-2 ratio, caspase-3/7 activity, and mitochondrial membrane permeability. CE also induces cytostasis in addition to apoptosis, substantiated by the reduced cytokinetic (replicative indices) and mitotic (mitotic indices and histone H3 Ser-10 phosphorylation) activities. Overall, the cellular GSH depletion and potential genotoxic effects by CE led the CHO cells to commit apoptosis and lowered cell division. The observed sensitivity of CHO cells doubts unintended adverse effects of CE on normal healthy cells, suggesting higher essentiality of further studies in order to establish its safety efficacy in therapeutic explorations.

Single, repeated dose toxicity and genotoxicity assessment of herb formula KIOM2012H

BackgroundTraditional medicine and herbal prescriptions are becoming more popular, and they account for a large share of the world’s healthcare research studies, developments, and market demands. Increasing scientific evidence of the substantive efficacies such as preventive health keeping pharmaceutical materials and dietary supplements can be found elsewhere. Above all, safety should be the critical premise for considering developmental materials such as pharmaceuticals without side effects and toxicity. MethodsThe authors formulated KIOM2012H (K2H) using four herbs that were reported to have medicinal effects—including anticancer, antiaging, antimicrobial, inflammation, and neuroprotective properties. In order to examine the toxicity, single and repeated dose toxicity, and genotoxicities of bacterial mutation, micronucleus, and chromosomal aberration assays were conducted. ResultsAll experimental observations and results showed normal findings. Toxicities or abnormal signs were not observed in all experimental assays, including oral administration, animal behavior, clinical findings, and changes in body weight in vivo. In vitro bacterial cultures produced no revertant colonies, and no increased numbers of structural or numerical aberrant metaphases were found in the metaphase chromosomes examined. Moreover, no significant increased frequency of micronucleus was observed in any of the doses used. Overall, no acute toxicity or genotoxicity was found in all analysis parameters in all the assays conducted. ConclusionReviewing the results as a whole, K2H extract was regarded as a safe material with no toxicity, and can be applied for the research and development of complementary and alternative medicines with improved efficacy in current therapeutic healthcare, based on traditional medicine and herb resources.

ДОЦІЛЬНІСТЬ ВИКОРИСТАННЯ ВІТАМІННИХ ХІМІОПРЕВЕНТЕРІВ ДЛЯ КОРЕГУВАННЯ ІНДУКЦІЇ ХРОМОСОМНИХ АБЕРАЦІЙ СИНТЕТИЧНИМ ХАРЧОВИМ АРОМАТИЗАТОРОМ «КАРАМЕЛЬ»

Flavors “Caramel” induced in root meristem cells of onion Allium cepa L. single and paired fragments of chromosome and dicentric chromosomes, which are the result of chromosomal aberrations such as deletions and translocations. It was found induction of dominant lethal mutations and unfertilized eggs by different concentration of flavors in Drosophila melanogaster test. As well as under flavors influence we have shown the induction of a wide range of chromosome aberrations in metaphase bone marrow cells of white mice Mus musculus L., which included not only chromosomes but also genomic mutations. This could be evidence of spindle apparatus disruption and chromosomes nondisjunction as an influence of mutagenic agents, which may include a flavoring component themselves as well as their metabolites appearing in the biotransformation in mammal’s organism. We observed a partial decrease of aberrant metaphases and dominant lethal mutations after vitamin A and C application.

Gross Chromosomal Aberrations are Early Alarms for Malignancy: A Re-Emphasis

A few chromatin dots of variable sizes were encountered in squashes of brain tumour tissues viz. medulloblastoma, ependymoma and tuberculoma along with hyper and hypoploid chromosome counts way back in 1973. Similar chromosomal features were again observed in metaphases of persons exposed to methylisocyanate gas in Bhopal during midnight of 2nd to 3rd December, 1984. Follow-up studies continued during 1984-1999 and comparative assessments on chromosomal damages were attempted by SCE (sister chromatid exchanges) as well as by scoring more than a dozen types of chromosomal aberrations on 678 individuals by C and G-banding and Feulgen’s and Aceto-orcein staining procedures on cultured-lymphocytes, details of which, have been published earlier. This short paper reemphasizes the importance of certain chromatin dots (named as Marker dots) which were seen emanating from chromosomes. These marker dots appeared reliable early indicators of neoplastic transformations. During follow-up studies on various malignancies we had recorded presence of these marker dots in almost all of them. This computation of slides involved careful scrutiny of more than 40,000 metaphases from patients of various malignancies, pathological disorders and newly discovered chromosome syndromes (e.g. Crossed Renal Ectopia with pelvic lipomatosis; Hemihypertrophy with melanosis of Ito, etc.). Intriguingly, only selective chromosomes are involved (chromosomes 1, 2, 3, 5, 6, 8, 9, 11, 12, 13, 16, 17 and Y) in emanating marker dots. Obviously, it appears that the molecular attenuation of chromatin structures movable from chromosomes is related with triggering neoplastic transformations. Specific attention was paid on appearance of “marker dots” which were observed in seemingly normal persons but later, after a gap of 2 to 6 years, some of them exhibited signs of malignancy. Such observations were possible only on those persons who could be re investigated after a gap of 2 or 3 years. Gross chromosomal aberrations such as PCD (premature centromeric divisions) acrocentric associations, hyperploid cells, translocations and deletions with marker dots appear to be precursors to firm installation of chromosomal mutagenesis in cultured lymphocytes. Obviously, search for marker dots and these aberrations in metaphases of person(s) belonging to a cancer-patient family can be of vital importance to warn for early diagnosis and prognostic approach. Marker dots simply “alarm” to inform firm installation of chromosomal mutagenesis which encompasses various chromosomal aberrations. In turn, due to many other intragenic factors, cells are transformed to malignant cells in certain individuals.

Isolate Loss of Y Chromosome Decreases the Risk of Leukemic Transformation in the Myelodysplastic Syndromes. a Study By the Spanish Group of Myelodysplastic Syndromes

▪BackgroundLoss of chromosome Y (-Y) is observed in 4-10% of male MDS patients as a single cytogenetic abnormality, and is associated to a better outcome. -Y is also known to be an age related phenomenon occurring in 8-10% of elderly men. The clinical significance of -Y in MDS has been a matter of discussion. However, there is increasing evidence that -Y involves the clonal population in MDS cases, and is more likely to be associated with a hematological malignancy when it involves a greater number of metaphases.AimsIn this study we aimed to 1) analyze the prognostic impact of -Y in the context of the IPSS-R cytogenetic classification, 2) evaluate the clinical significance of the percentage of metaphases with isolated -Y, 3) test whether finding -Y may predispose to over diagnose MDS in patients with borderline morphological features.MethodsWe evaluated 3581 male patients from the Spanish Registry of MDS with a diagnosis of MDS or CMML (WHO 2001). Patients with isolated -Y and normal karyotype by conventional cytogenetic analysis were selected. The main study outcomes were survival from diagnosis and transformation into acute myeloid leukemia (AML). Survival curves were drawn using the Kaplan-Meyer method and compared by log-rank test. The cumulative incidence of AML was estimated by taking non AML-related death as a competing risk. Statistical comparisons were done by the Mann-Whitney U-test for continuous data, and the chi-square test for categorical factors. Stata, version 11, software (www.stata.org) was used for the statistical analysis.ResultsIsolated -Y was identified in 177 patients (-Y group) and compared with the 2246 male patients with normal karyotype (46,XY group). The median age for the whole series was 74 years old (IQR 67-80). -Y patients were found to be older than patients in the 46,XY group with a median (IQR) of 78 (74-83) versus 74 (66-79) years old, respectively (n=0.0001). There was no difference between both groups in terms of hemoglobin concentration, neutrophil and platelet counts at diagnosis. Percentage of bone marrow (BM) blasts was significantly lower in the -Y group (median (IQR): 2(0-3) vs. 2(1-5), respectively; p=0.003). Differences in distribution by WHO 2001 classification of MDS were not found between the two groups. After a follow-up of 2,190 patient-years, 1684 (69.5%) patients had died and the median actuarial survival was 3.6 years (95% C.I, 3.3-3.9). A trend towards a larger median survival in the -Y group versus 46,XY group was observed, since it not reached statistical significance (5.2 years [95% CI, 3.89 to 6.40] versus 4.26 years [95% CI, 3.89 to 4.59], respectively) (P=0. 17; Fig. 1A). After a median follow-up of 1.6 years (IQR: 0.8 - 3.7) from MDS diagnosis, 296 patients had progressed to AML, 901 had died without AML, and 1224 were censored alive without transformation. -Y was associated with a decreased incidence of AML at univariate analysis (figure 1B), and after adjustment for the percentage of BM blasts (SHR: 0.46; 95%CI: 0.24-0.88; p=0.02). Within the -Y group, neither survival nor the risk of leukemic transformation were influenced by the percentage of aberrant metaphases (>75% vs, ≤ 75%). From the whole series, only 6.4% of the cases were classified as not having a "MDS strong phenotype", defined by the presence of megakaryocytic dysplasia, more than 5% blasts in the bone marrow, or more than 15% ring sideroblasts. These cases were uniformly distributed between the two groups, suggesting that the better outcome in the -Y group cannot be explained by enrichment in cases misdiagnosed as MDS.ConclusionsOur results derived from the largest series of patients with loss of chromosome Y support the current recommendation of classifying patients with -Y within the very good risk category of the IPSS-R for MDS and rule out a selection bias as a possible explanation of this better outcome. An analysis of the molecular basis of MDS with isolated -Y would be of interest as it may provide a biological basis of protection against AML progression. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

RESIDENTS OF INDUSTRIAL REGIONS: LONG TERM DYNAMICS OF SPONTANEOUS MUTAGENESIS

The studies shows results of the 10-years research related to the frequency of chromosome aberrations in peripheral blood lymphocytes of North Ossetia inhabitants. The study of cytogenetic indexes in the blood of healthy individuals showed an increase in the average frequency of chromosome aberrations as time passed. The long-term cytogenetic study of North Ossetia residents’ blood showed the growth of medium frequency of aberrant metaphases from 2.07 ± 0.37 to 3.04 ± 0.16 in the group of adults and from 0.5 ± 0.25 to 1.9 ± 0.2 in the groups of children. The most significant increase among all studied cytogenetic indexes in groups of North Ossetia adults and children was in 2009 due to accidental release at one of the metallurgical enterprises in the capital city. All studied groups were characterized by prevailed acentric fragments among chromosome disorders, with an increase of all types of aberrations as time passed. There was a growth in the proportion of chromosome aberrations (adults had an increase of double fragments from 0.20 ± 0.11 to 0.60 ± 0.15 and chromosome exchanges from 0.27 ± 0.15 to 0.64 ± 0.13, children: from 0.13 ± 0.13 to 0.26 ± 0.12 and from 0 to 0.26 ± 0.20 respectively). Sex and smoking factors had no significant effect on the studied samples during the observation period. The 10-year (2002-2011) dynamics analysis of cytogenetic disorders has shown an intensification of mutagenic effects on residents of the republic, which were the most severe after accidental releases in 2009. The results of cytogenetic studies suggest that a mutagenic impact of chemical and radioactive nature takes place.

THE QUANTITY OF CELLS NECESSARY FOR THE ANALYSIS OF CHROMOSOMAL ABERRATIONS AT RESIDENTS OF KUZBASS PATIENTS WITH CANCER OF LUNG

The pilot research executed in 2014 in selection of patients with lung cancer (LC) and healthy residents of Kemerovo region (338 Caucasians: 159 having LC and 179 healthy) has shown statistically significant distinction between groups of level and range of chromosome aberrations in blood lymphocytes. Patients of cancer detection centre (before treatment) had increased chromosome aberration values, both of chromatid and chromosomal types. 250 metaphases were analyzed for each individual. For accuracy test of the received quantitative characteristics of mutational process and definition required and enough number of cells necessary for the analysis, at essential increase in further selection volume, 50 LC patients (selected randomly), comparison of cytogenetic data has been carried out in the analysis of 200 cells, then 400, 600, 800, 1000 and over 1000 metaphases. Totally, in this subgroup 50 000 cells which were at a mitosis metaphase stage have been studied. Statistical data processing was carried out with the use of a software package for Windows Statistica 8.0. Using Mann-Whitney U-criterion it is confirmed that frequency of aberrant metaphases doesn't change significantly for statistics in case of increase in analyzed cells quantity; the analysis of 200 cells gives no less reliable information about the individual CA level, than the analysis of 1000 and more cells. In this regard it is possible to make the conclusion that 200 cells is necessary and enough number of analysed metaphases at the assessment of individual level and chromosomal aberrations' range for Kemerovo region residents who have lung cancer.

Application of a Short Tandem Repeat Based PCR Assay for Chronological Monitoring of Myelodysplastic Syndrome (MDS) Patients with Deletion of Chromosome 5q Following Lenalidomide Treatment

IntroductionDeletion of chromosome 5q (del(5q)) defines a distinct clinical subtype of myelodysplastic syndromes (MDS) and qualifies patients to specific treatment with Lenalidomide (LEN). Therefore, detection and monitoring of this deletion is an important element in routine clinical diagnostics for determining molecular response. Current methodologies for performing these analyses consist of cytogenetics, fluorescence in situ hybridization (FISH) or microarrays. All of these methods have downsides due to the high demands to the input material, i.e. viable cells or necessity for large amounts of high quality genomic DNA (gDNA). To perform quantitative assessment of cytogenetic lesions in low quantity or residual material we here present the establishment of a PCR-based assay for interrogation of del(5q) in MDS, based on the allelic loss at heterozygous short tandem repeat (STR) loci within deleted regions.MethodsGenomic DNA was isolated from bone marrow (BM) and peripheral blood (PB) of n=86 MDS del(5q) patients. 49 non-del(5q) MDS patients were used as controls. Serial chronological BM samples (n=95) following treatment with LEN from n=40 del(5q) patients, who were enrolled in the LEMON-5 trial from the German MDS study group, were analysed. Using 10ng DNA, 12 fluorochrome-labelled PCR amplicons of STR loci located between chromosomal bands 5q21 and 5q31 were amplified in a single optimized multiplex-PCR reaction. Subsequently, amplicon fragment analysis was carried out via capillary electrophoresis and allele size quantification of heterozygous STR loci was performed. Finally, the degree of skewing in the allelic ratios of all informative STR markers was averaged and translated into an allelic burden of del(5q).ResultsPaired quantitative correlation of clone sizes using STR-PCR and interphase FISH was carried out in n=34 samples and revealed highly concordant results with r²=0.924. The diagnostic accuracy of the PCR assay was evaluated by receiver operating characteristic (ROC) analysis and revealed an area under the curve of 0.989 (sensitivity and specificity of 0.977 and 0.948, respectively). Prior to treatment with LEN, clone sizes as determined by STR-PCR were heterogeneous (mean: 57%, range: 11-91%). During follow-up analysis, while cytogenetic analyses failed (e.g. metaphase failure) in 7/40 (18%) cases, our STR-PCR assay successfully generated estimates of del(5q) cell burden in all available samples. Upon LEN treatment, n=12 patients achieved major cytogenetic remission (absence of del(5q)-positive metaphases). The mean clone size carrying del(5q) determined by STR-PCR in that group was 7% (range 3 - 10%) and significantly increased compared with n=15 patients who reached minor cytogenetic response (defined as 50% reduced aberrant metaphases, mean 13%, range 5 - 39%, p=0.025). Intriguingly none of n=6 patients without cytogenetic response achieved a del(5q) clone size of less than 35% as determined by STR-PCR (mean 46%, range 35 - 66%), highlighting the correlation of PCR based follow-up analysis with currently used cytogenetic methods for response evaluation. Finally in n=93 matched PB and BM samples a correlation of del(5q)-frequency in BM versus PB showed r²=0.81. Moreover, in 96% of samples in which the BM still showed clone sizes >10%, we reliably detected del(5q) in corresponding PB cells with a robust sensitivity of 5% deleted cells.DiscussionWe present a highly adaptable tool for precise measurement of large chromosomal deletions, requiring only minute amounts of genomic DNA. It shows a very good quantitative correlation with established methods and good diagnostic accuracy. Most importantly, this PCR based assay does not require dividing cells so it can be performed from PB, which shows a sufficient correlation with clone sizes in BM and rarely involves the risk of underrepresentation of del(5q)-clones in PB, possibly allowing the use of PB as a regular specimen for clone size monitoring. Thus, especially in the context of serially monitored patients this assay represents an alternative method for less invasive tracking of cytogenetically aberrant clones. DisclosuresPlatzbecker:Celgene: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Boehringer: Research Funding. Schlenk:Janssen: Membership on an entity's Board of Directors or advisory committees; Pfizer: Honoraria, Research Funding; Boehringer-Ingelheim: Honoraria; Teva: Honoraria, Research Funding; Arog: Honoraria, Research Funding; Novartis: Honoraria, Research Funding; Daiichi Sankyo: Membership on an entity's Board of Directors or advisory committees. Bug:TEVA Oncology, Astellas: Other: Travel Grant; NordMedica, Boehringer Ingelheim, Gilead: Membership on an entity's Board of Directors or advisory committees; Celgene, Novartis: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Evaluation of in vivo cytogenetic toxicity of europium hydroxide nanorods (EHNs) in male and female Swiss albino mice

Our group already demonstrated that europium hydroxide nanorods (EHNs) show none or mild toxicity in C57BL/6 mice even at high dose and exhibited excellent pro-angiogenic activity towards in vitro and in vivo models. In the present study, we evaluated the in vivo cytogenetic toxicity of intraperitoneally administered EHNs (12.5–250 mg/kg/b.w.) in male and female Swiss albino mice by analyzing chromosomal aberrations (CAs), mitotic index (MI), micronucleus (MN) from bone marrow and peripheral blood. Furthermore, we performed the cytogenetic toxicity study of EHNs towards Chinese hamster ovary (CHO) cells, in order to compare with the in vivo results. The results of CA assay of mice treated with EHNs (12.5–125 mg/kg/b.w.) showed no significant change in the formation of aberrant metaphases compared to the control group. Also, there was no significant difference in the number of dividing cells between the control group and EHNs-treated groups observed by MI study, suggesting the non-cytotoxicity of EHNs. Additionally, FACS study revealed that EHNs do not arrest cells at any phase of cell cycle in the mouse model. Furthermore, MN test of both bone marrow and peripheral blood showed no significant differences in the induction of MNs when compared with the control group. In vitro results from CHO cells also support our in vivo observations. Considering the role of angiogenesis by EHNs and the absence of its genotoxicity in mouse model, we strongly believe the future application of EHNs in treating various diseases, where angiogenesis plays an important role such as cardiovascular diseases, ischemic diseases and wound healing.

Tumor treating fields perturb the localization of septins and cause aberrant mitotic exit.

The anti-tumor effects of chemotherapy and radiation are thought to be mediated by triggering G1/S or G2/M cell cycle checkpoints, while spindle poisons, such as paclitaxel, block metaphase exit by initiating the spindle assembly checkpoint. In contrast, we have found that 150 kilohertz (kHz) alternating electric fields, also known as Tumor Treating Fields (TTFields), perturbed cells at the transition from metaphase to anaphase. Cells exposed to the TTFields during mitosis showed normal progression to this point, but exhibited uncontrolled membrane blebbing that coincided with metaphase exit. The ability of such alternating electric fields to affect cellular physiology is likely to be dependent on their interactions with proteins possessing high dipole moments. The mitotic Septin complex consisting of Septin 2, 6 and 7, possesses a high calculated dipole moment of 2711 Debyes (D) and plays a central role in positioning the cytokinetic cleavage furrow, and governing its contraction during ingression. We showed that during anaphase, TTFields inhibited Septin localization to the anaphase spindle midline and cytokinetic furrow, as well as its association with microtubules during cell attachment and spreading on fibronectin. After aberrant metaphase exit as a consequence of TTFields exposure, cells exhibited aberrant nuclear architecture and signs of cellular stress including an overall decrease in cellular proliferation, followed by apoptosis that was strongly influenced by the p53 mutational status. Thus, TTFields are able to diminish cell proliferation by specifically perturbing key proteins involved in cell division, leading to mitotic catastrophe and subsequent cell death.

Automation of the in vitro micronucleus and chromosome aberration assay for the assessment of the genotoxicity of the particulate and gas–vapor phase of cigarette smoke

Total particulate matter (TPM) and the gas–vapor phase (GVP) of mainstream smoke from the Reference Cigarette 3R4F were assayed in the cytokinesis-block in vitro micronucleus (MN) assay and the in vitro chromosome aberration (CA) assay, both using V79-4 Chinese hamster lung fibroblasts exposed for up to 24 h. The Metafer image analysis platform was adapted resulting in a fully automated evaluation system of the MN assay for the detection, identification and reporting of cells with micronuclei together with the determination of the cytokinesis-block proliferation index (CBPI) to quantify the treatment-related cytotoxicity. In the CA assay, the same platform was used to identify, map and retrieve metaphases for a subsequent CA evaluation by a trained evaluator. In both the assays, TPM and GVP provoked a significant genotoxic effect: up to 6-fold more micronucleated target cells than in the negative control and up to 10-fold increases in aberrant metaphases. Data variability was lower in the automated version of the MN assay than in the non-automated. It can be estimated that two test substances that differ in their genotoxicity by approximately 30% can statistically be distinguished in the automated MN and CA assays. Time savings, based on man hours, due to the automation were approximately 70% in the MN and 25% in the CA assays. The turn-around time of the evaluation phase could be shortened by 35 and 50%, respectively. Although only cigarette smoke-derived test material has been applied, the technical improvements should be of value for other test substances.

Genotoxicity of single-walled carbon nanotubes: in vitro study on human embryonic fibroblast cells.

The effects of single-walled carbon nanotubes on the levels of DNA aberrations, chromosome and genome disorders were studied on human embryonic fibroblasts, their karyotype was analyzed by the spectral karyotyping method. The level of DNA aberrations increased after 3-h exposure to the nanotubes. No appreciable increase in the incidence of aberrant metaphases, micronuclei, and chromosome 1, 6, 8, 11, X, and Y aneuploidy after 24- and 48-h incubation with the nanotubes were detected.

Interaction of DNA-lesions induced by sodium fluoride and radiation and its influence in apoptotic induction in cancer cell lines

Fluoride is an essential trace element but also an environmental contaminant with major sources of exposure being drinking water, food and pesticides. Previous studies showed that sodium fluoride (NaF) at 5mM or more is required to induce apoptosis and chromosome aberrations and proposed that DNA damage and apoptosis play an important role in toxicity of excessive fluoride. The aim of this study is directed to understand the nature of DNA-lesions induced by NaF by allowing its interaction with radiation induced DNA-lesions. NaF 5mM was used after observing inability to induce DNA damages and apoptosis by single exposure with 50μM or 1mM NaF. Co-exposure to NaF and radiation significantly increased the frequency of aberrant metaphases and exchange aberrations in human lymphocytes and arrested the cells in G1 stage instead of apoptotic death. Flow cytometric analysis, DNA fragmentation and PARP-cleavage analysis clearly indicated that 5mM NaF together with radiation (1Gy) induced apoptosis in both U87 and K562 cells due to down regulation of expression of anti-apoptotic proteins, like Bcl2 in U87 and inhibitors of apoptotic proteins like survivin and cIAP in K562 cells. This study herein suggested that single exposure with extremely low concentration of NaF unable to induce DNA lesions whereas higher concentration induced DNA lesions interact with the radiation-induced DNA lesions. Both are probably repaired rapidly thus showed increased interactive effect. Coexposure to NaF and radiation induces more apoptosis in cancer cell lines which could be due to increased exchange aberrations through lesions interaction and downregulating anti-apoptotic genes.

In CML Patients with Good Response to TKIs Other Gene Mutations Are Frequently (37%) Present in Addition to Philadelphia Negative, Cytogenetically Aberrant Clones but Are Rare (4%) in Cases with MMR and Normal Karyotype

Background: In chronic myeloid leukemia (CML) clonal chromosome aberrations in metaphases not carrying a t(9;22)(q34;q11) have been described during treatment with tyrosine kinase inhibitors (TKI), so-called Philadelphia-negative (Ph-) clones. Very rarely transformation to MDS was observed in patients carrying such Ph- clones but mainly restricted to patients harboring -7. Overall, the clinical significance of this phenomenon remains obscure.Aim: 1) Analyze in a large cohort of TKI-treated CML patients who developed Ph- clones the presence and occurrence of molecular mutations over time. 2) Evaluate whether molecular mutations are also present in CML patients who were at least in major molecular remission (MMR) and presented with a normal karyotype.Patients and Methods: First Cohort: 51 CML patients (pts, 23 males, 28 females; median age: 60 yrs, range: 37-84 yrs) with response to TKI (imatinib only: n=32, nilotinib only: n=2, imatinib and dasatinib or nilotinib: n=11, all three TKIs: n=6) who developed Ph- clones. Cytogenetics in these pts were as follows: +8 sole (n=24), -Y (n=8), -7 sole (n=4), +9 (n=2), other trisomies (n=4), 9 had other aberrations including some with combinations of two different clones (n=4). In median these abnormalities were present in 30% (range 8-100%) of analyzed metaphases. BCR-ABL1 levels at the time point of analysis were between 0 and 3.8 (median: 0.023) according to international scale. Second Cohort: 50 CML pts (24 males, 26 females; median age: 56 yrs, range: 21-83 yrs), who were at least in MMR and without development of any cytogenetic aberration after 3 years of imatinib treatment. Median time from start of therapy to analysis was 2.6 years (range 3 months to 14 yrs).All cases were analyzed with a pan-myeloid gene panel of 29 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, NOTCH1, NPM1, NRAS, PRPF40B, PTPN11, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2 and ZRSR2. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Billerica, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). In addition, RUNX1 was sequenced on the 454 NGS platform (454 Life Sciences, Branford, CT).Results: In the first cohort 28 mutations were found in 19 patients, as 5 patients had 2 and 2 patients even 3 mutations.Thus,in 19/51 pts (37.3%) ≥1 mutation was detected. Median mutation load was 11.5% (range: 2-56%). In detail, mutations in the following genes were detected: ASXL1 (n=9), DNMT3A (n=7), RUNX1 (n=3), NRAS (n=2), TET2 (n=2) and one each in CBL, EZH2, IHD1, PRPF40B, and TP53. Subsequently, these mutations were evaluated in samples from earlier or later time points (18 pts with a total of 235 samples, range: 3-20 samples/pt). In 12 cases a sample from diagnosis of CML was available. In 2 cases a CBL and an ASXL1 mutation were already detectable at low levels, 1.4% and 2%, respectively, at the time of diagnosis and later increased with decreasing BCR-ABL1 levels. In all other 10 cases the mutations were not detectable at diagnosis and were for the first time detectable during TKI treatment (in median after 24 months after diagnosis, range 2-73 months). In the remaining 6 cases date of occurrence could not be determined by backtracking as all earlier samples available were positive for the respective mutation. However, the over time mutation levels were inversely related to BCR-ABL1 expression indicating the presence in independent clones.Within the second cohort with cases in MMR that remained cytogenetically normal only in 2 of the 50 pts (4%) mutations were detected. In one patient a DNMT3A mutation was detected that could be monitored for 8 years with constant low mutation load (3-6%). This was not detectable at diagnosis and occurred after 6 months on imatinib. Very similarly, in the second case a TET2 mutation was first detected after 6 months on imatinib with a mutation load of 2% that very slowly increased to 7% within 8 years.Conclusions: 1) In CML patients that develop Ph- clones other mutations occur in 37.3%. 2) In contrast, in randomly selected CML pts with MMR that are cytogenetically normal, molecular mutations can be detected in only 4%. 3) The clinical importance of molecular mutations in CML in MMR remains unclear. 4) However, these results implicate that chromosomal aberrations are an indicator for genomic instability, also at the molecular level. DisclosuresSchnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Acute toxicity and genotoxicity study of fermented traditional herb formula Guibi-tang

Ethnopharmacology relevanceGuibi-tang (Guipi-tang in Chinese and Kihi-to in Japanese) is a multi-herb traditional medicine commonly prescribed to treat psychoneurosis in East Asia. Although this medicine has been widely used, there is little available information on the safety and toxicity of Guibi-tang, especially on the fermented one. Materials and methodsGuibi-tang, composed of 12 herbs, was fermented with bacteria and lyophilized. Single dose acute toxicity in rats was observed for 14 days after administration. Genetic toxicity of fermented Guibi-tang was evaluated on bacterial reverse mutation in Salmonella and Escherichia spp., chromosome aberrations in Chinese hamster ovary cells, and micronucleus formation in mice. Ingredients in FGBT were identified and quantified by high performance liquid chromatography–mass spectrometry. ResultsIn acute oral toxicity study, behavior, clinical signs and body weight changes were normal observing in all experimental animals. No revertant colonies were found in any bacterial cultures examined. Morphological or numerical anomalies and significant increased number of aberrant metaphases were not observed. Micronucleus assay showed no significant increases in the frequency of inducing micronuclei in any dose examined. Decursinol, decursin, glycyrrhizin, and 6-gingerol in fermented Guibi-tang were identified and quantitated. As a whole, no acute and genotoxic effects were found in all the assays and parameters analyzed. ConclusionFermented Guibi-tang was recognized as safe and non-toxic, and therefore can be used for applications of traditional medicine in modern complementary and alternative therapeutics and health care.

Hyparrhenia hirta: A potential protective agent against hematotoxicity and genotoxicity of sodium nitrate in adult rats.

The present study was carried out to examine the adverse hematotoxic and genotoxic effects of water nitrate pollution on male adult rats and the use of hyparrhenia hirta methanolic extract in alleviating these effects. Sodium nitrate (NaNO3 ) was administered to adult rats by oral gavage at a dose of 400 mg kg(-1) bw daily for 50 days, while hyparrhenia hirta methanolic extract was given by drinking water at a dose of 1.5 mg mL(-1) (200 mg kg(-1) bw). The NaNO3 -treated group showed a significant decrease in red blood cell count, hemoglobin and hematocrit and a significant increase in total white blood cell, in neutrophil and eosinophil counts. Platelet count, mean corpuscular volume, mean corpuscular hemoglobin, and mean corpuscular hemoglobin concentration remained unchanged in treated groups compared to those of controls. Meanwhile, the results showed a marked reduction in the antioxidant enzyme activities, such as superoxide dismutase, catalase, and glutathione peroxidase, along with an elevation in the level of lipid peroxidation and a reduction in the total glutathione content, indicating the induction of oxidative stress in the erythrocytes of NaNO3 -treated group. Interestingly, NaNO3 treatment showed a significant increase in the frequencies of total chromosomal aberrations, aberrant metaphases and micronucleus in bone-marrow cells. The oxidative stress induced by nitrate treatment might be the major cause for chromosomal rearrangements as free radicals leading to DNA damage. Hyparrhenia hirta methanolic extract appeared to be effective against hematotoxic and genotoxic changes induced by nitrate, as evidenced by the improvement of the markers cited above.

POTENTIAL ANTICLASTOGENIC EFFECT OF HYPERFORIN

Summary Hyperforin is a prenylated phloroglucinol derivative which occurs in the plant Hypericum perforatum L. It has several medicinally important properties (antidepressant, anti-inflammatory, proapoptotic, antibacterial and antiangiogenic). To enable its medicinal use, it is necessary to investigate its potential genotoxic effect on human cell lines. We also observed an anticlastogenic effect of hyperforin towards the indirect mutagen benzo(a)pyrene. Benzo(a)pyrene is a widespread polycyclic aromatic hydrocarbon. We performed experiments on human tumor cell line HepG2, using the in vitro mammalian chromosome aberration test. We investigated two cytogenic parameters: the number of aberrant metaphases and the total number of chromosome aberrations. We found out that hyperforin was not genotoxic on human cell line HepG2. The second part of our results implies that hyperforin had an anticlastogenic effect against the indirect mutagen benzo(a)pyrene in our experimental conditions. In future we will continue our research by using another range of hyperforin concentrations, other cell lines and other chemical mutagens.

Terminalia arjuna, a herbal remedy against environmental carcinogenicity: An in vitro and in vivo study

BackgroundMedicinal plants have been a major source of therapeutic agents from ancient times to cure diseases. The evaluation of rich heritage of traditional medicine is essential. The bark of Terminalia arjuna is rich in polyphenols (60–70%) including flavonoids and tannins. AimThe aim of the present investigation is to highlight the anticarcinogenic and antimutagenic potential of extracts of T. arjuna. Subject and methodsIn this experiment we have used human lymphocyte culture and bone marrow cells of albino mice as assay system. The parameters studied included chromosomal aberrations (CA), sister chromatid exchanges (SCEs) and cell growth kinetics (RI) both in the presence and in the absence of exogenous metabolic activation system for in vitro experiment, whereas total aberrant cells and the total frequencies of aberrations were taken for in vivo study. ResultsThe role of T. arjuna extracts in reducing metaphase aberrations due to aflatoxin B1 is quite significant, the reduction varying from 23.49%, 42.47%, and 59.65% down to 12.32%, 28.00%, and 36.88% respectively at the highest dose (TA4) for the three different durations viz., 24, 48 and 72h. Similarly the number of sister chromatid exchanges got reduced from a higher level of 15.00±1.40 per cell to 7.70±0.50 per cell with S9 mix at 48h of treatment. The replication index was enhanced from 1.33 to 1.55 in vitro. Similar trends were noticed in the in vivo experiments i.e., effective reductions in clastogeny ranging from 15.22% to 54.82% from the mutagen treated positive control and the total frequencies in aberrant cells got reduced from 429 due to AFB1 to 141 due to 5th concentration of Terminalia extracts at 32h of exposure. ConclusionThe ameliorating potential of Terminalia extracts was dose and time dependant.

Tyrosine Kinase Inhibitor Treated CML Patients Harboring Philadelphia-Negative Cytogenetically Aberrant Clones Show Molecular Mutations In 31% Of Cases Not Present At Diagnosis: A High-Throughput Amplicon Sequencing Study Of 29 Genes

BackgroundIn chronic myeloid leukemia (CML), clonal chromosome aberrations in metaphases not carrying a t(9;22)(q34;q11) have been described during treatment with tyrosine kinase inhibitors (TKIs), so-called Philadelphia-negative (Ph-) clones. The most frequent abnormalities in these Ph- clones reported are -7, +8 and -Y. This pattern of cytogenetic aberrations is comparable to alterations found in MDS. In the majority of patients no clear evidence of a secondary hematologic malignancy is observed. However, few cases especially with -7 were reported to evolve to MDS or even AML. Aim1) Analyze whether TKI-treated CML patients who developed Ph- clones also harbour molecular mutations. 2) Evaluate whether these mutations were already present at diagnosis of CML. Patients and MethodsWe selected 42 patients (pts) treated with TKIs (Imatinib: n=27, Nilotinib: n=2, and 13 pts who were treated with 2 or 3 different TKIs) who developed Ph- clones. Median time from start of therapy to analysis was 3.2 years (range 8.9 months-13.5 years). 23 cases were male and 19 female; median age was 63.3 years (range: 34.1-83.9 years). Cytogenetics in pts with Ph- clones were as follows: +8 sole (n=18), -Y (n=7), -7 sole (n=2), +9 (n=2), an 10 cases with various abnormalities. Additional three cases had two different clones: 1) -7 and +8; 2) +8 and +Y, 3) -Y and +Y. Median clone size was 37.5% (range 10-100%) of all metaphases. BCR-ABL1 levels at the time point of analysis were between 0 and 4.5 (median: 0.019) according to international scale. All cases were analyzed with a pan-myeloid gene panel of 29 genes: ASXL1, BCOR, BRAF, CBL, DNMT3A, ETV6, EZH2, FLT3 (TKD), IDH1, IDH2, JAK2, KIT, KRAS, MLL-PTD, NOTCH1, NPM1, NRAS, PRPF40B, PTPN11, RUNX1, SF1, SF3A1, SF3B1, SRSF2, TET2, TP53, U2AF1, U2AF2 and ZRSR2. Either complete coding genes or hotspots were first amplified by a microdroplet-based assay (RainDance, Lexington, MA) and subsequently sequenced with a MiSeq instrument (Illumina, San Diego, CA). RUNX1 was sequenced on the 454 Life Sciences NGS platform and the MLL-PTD was analyzed by quantitative real time PCR. Median coverage per amplicon was 2215 reads (range 100-24,716). The lower limit of detection was set at a cut-off of 1.5%. ResultsIn total, in 13/42 pts (31.0%) one (n=9) or two (n=4) of the analyzed genes were mutated. Median mutation load was 15% (range: 3-50%). In detail, mutations in the following genes were detected: ASXL1 (n=5), DNMT3A (n=4), NRAS (n=2) and each one in BCOR, CBL, MLL-PTD, RUNX1, TET2 and ZRSR2. 4 pts had combinations of: 2 ASXL1mut, 2 NRASmut, BCORmut and RUNX1mut, or ASXL1mut with MLL-PTD, respectively. Subsequently, the mutations were tracked as far as serially collected samples were available (12 pts with a total of 125 samples, range: 3-20 samples/pt) from earlier or later time points. In 6 cases it clearly could be shown that the mutations were not present at diagnosis and arose 5 months, 7 months, 14 months, 15 months, 5 years, and 7 years after start of TKI therapy, respectively. In all these cases the mutation loads increased in parallel to decreasing BCR-ABL1 levels. In one of these cases one mutation was found 3 years and a second 11 years after start of therapy. Importantly, in three of these cases the molecular mutations were detectable before the cytogenetic aberration in the Ph- clone. In six further cases mutations were present with similar levels in several samples but due to lack of samples from initial diagnosis no estimation of the time to their occurrence was possible. In one of these cases the BCR-ABL1 load increased again during therapy and the DNMT3A mutation decreased showing that the BCR-ABL+ clone and the DNMT3Amut clone were competitive clones. Conclusions1) For the first time we show that more than 30% of CML patients treated with TKI who develop Ph- clones as defined by cytogenetic abnormalities, also harbour molecular mutations. 2) For most of the cases it was shown that these mutations arose during treatment. 3) In some cases the molecular mutations were detectable before the cytogenetic aberrations. This may be regarded as further proof of the malignant character of these Ph- clones. However, none of the analyzed cases, so far, developed a secondary disease, i.e. MDS or AML, thus the relevance of these findings is still unclear and needs further attention. Disclosures:Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kuznia:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Jeromin:MLL Munich Leukemia Laboratory: Employment. Alpermann:MLL Munich Leukemia Laboratory: Employment. Roller:MLL Munich Leukemia Laboratory: Employment. Albuquerque:MLL Munich Leukemia Laboratory: Employment. Weissmann:MLL Munich Leukemia Laboratory: Employment. Eder:MLL Munich Leukemia Laboratory: Employment. Dicker:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Kohlmann:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership; Novartis: Research Funding. Hochhaus:Novartis: Research Funding. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Impact Of The Proportion Of Metaphases With Isolated Del(5q) On Clinical Outcomes In Lenalidomide (LEN)-Treated Patients With IPSS Low-/Int-1-Risk Myelodysplastic Syndromes (MDS) In MDS-003 and MDS-004

IntroductionThe proportion of aberrant metaphases is prognostic for overall survival (OS) in MDS patients with trisomy 8 (Mallo M, et al. Leuk Res. 2011;35:834-6). The impact of the proportion of metaphases with del(5q) on clinical outcomes, including OS, disease progression and response to therapy with LEN in MDS remains undefined. In two large multicenter studies of LEN (MDS-003 and MDS-004) in RBC transfusion-dependent patients with International Prognostic Scoring System (IPSS) Low- or Intermediate (Int)-1-risk del(5q) MDS, RBC transfusion independence (TI) ≥ 8 weeks was achieved in 51–67% of patients (List A, et al. N Engl J Med. 2006;355:1456-65; Fenaux P, et al. Blood. 2011;118:3765-76). This retrospective analysis evaluated response to treatment, progression to acute myeloid leukemia (AML) and OS by proportion of del(5q) metaphases in patients with isolated del(5q) from the MDS-003 and 004 studies. MethodsIn order to allow sufficient patient numbers for analysis, ≥ 16 metaphases were evaluated for del(5q) by standard karyotyping (MDS-003 and MDS-004) and 200 interphase nuclei were evaluated by fluorescence in situ hybridization (FISH; MDS-004 only) using a probe for the commonly deleted region 5q31 (LSI EGR1/D5S721, Abbott, Wiesbaden, Germany). Patients received LEN on days 1–21 of each 28-day cycle (10 mg) or continuously (5 mg or 10 mg), or placebo. In MDS-004, patients randomized to placebo could cross over to LEN 5 mg by week 16. RBC-TI ≥ 26 weeks, time to AML progression and OS were analyzed by the proportion of del(5q) metaphases or interphases (≤ 60% vs > 60%) using standard karyotyping and FISH, respectively. ResultsOf the 353 patients from MDS-003 and MDS-004, 194 had isolated del(5q) by standard karyotyping; median proportion of del(5q) metaphases was 96% (range 4–100). Baseline characteristics including age, time from diagnosis, RBC transfusion burden, hemoglobin level, platelet and absolute neutrophil counts were comparable among patients with ≤ 60% (n = 21) and > 60% (n = 173) del(5q) metaphases. Rates of RBC-TI ≥ 26 weeks were similar across patients in the ≤ 60% and > 60% groups (P = 0.6515). Time to AML progression was comparable for patients in the ≤ 60% group versus the > 60% group (log-rank test P = 0.9802); 2-year rates were 22.2% (95% confidence interval [CI]: 7.7–54.5%) and 14.6% (95% CI: 9.9–21.2%), respectively. Time to AML progression was similar when analyzed with death without AML as competing risk (Gray's test P = 0.5514). OS was longer in the > 60% versus the ≤ 60% group (log-rank test P = 0.0436); median OS was 3.7 years (95% CI: 3.0–4.2) and 2.4 years (95% CI: 1.5–4.9), respectively. In MDS-004, the proportion of del(5q) interphases was analyzed using FISH in 106 patients, including 46 with ≤ 60% and 60 with > 60%. When analyzed by FISH, rates of RBC-TI ≥ 26 weeks were similar across patients in the ≤ 60% and > 60% groups (P = 1.000). Time to AML progression and OS were similar across these groups (log-rank test P = 0.7311 and P = 0.8639, respectively) when analyzed by FISH. In the ≤ 60% and > 60% groups respectively, 2-year AML progression rates were 14.8% (95% CI: 6.9–30.1%) and 18.6% (95% CI: 10.4–32.0%), and median OS was 3.1 years (95% CI: 2.3–4.8) and 2.9 years (95% CI: 2.3–4.4). Time to AML progression was similar when analyzed with death without AML as competing risk (Gray's test P = 0.8631). ConclusionsIn IPSS Low- or Int-1-risk MDS patients with isolated del(5q) treated with LEN in MDS-003 and MDS-004 studies, baseline characteristics, RBC-TI ≥ 26 weeks and AML progression were comparable in patients with > 60% versus ≤ 60% del(5q) metaphases. Although similar across groups when analyzed by FISH in a subset of patients, surprisingly, OS was longer in patients with > 60% del(5q) metaphases than in those with ≤ 60% del(5q) metaphases by standard karyotyping. However, the number of patients with ≤ 60% del(5q) metaphases was limited and no adjustment was made for multiple testing. These findings suggest that the number of cells with the isolated del(5q) abnormality measured by FISH does not impact clinical outcome in this RBC transfusion-dependent study population, but this finding could not be confirmed for OS by standard karyotyping. Disclosures:Giagounidis:Celgene: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees. List:Celgene: Serve on Celgene Data Safety & Monitoring Committee Other. Hellström-Lindberg:Celgene: Membership on an entity's Board of Directors or advisory committees, Research Funding. Sekeres:Celgene: Membership on an entity's Board of Directors or advisory committees; Amgen: Membership on an entity's Board of Directors or advisory committees. Mufti:Celgene: Honoraria, Membership on an entity's Board of Directors or advisory committees, Research Funding. Schlegelberger:Celgene: Consultancy. Morrill:Celgene: Employment, Equity Ownership. Wu:Celgene: Employment, Equity Ownership. Skikne:Celgene: Employment, Equity Ownership. Fenaux:Celgene: Honoraria.