Abnormal Metaphases Research Articles

Molecular cytogenetic screening of sex chromosome abnormalities in young horse populations.

Chromosomal abnormalities occur in the equine population at a rate of approximately 2%. The use of molecular cytogenetic techniques allows a more accurate identification of chromosomal abnormalities, especially those with a low rate of abnormal metaphases, demonstrating that the actual incidence in equine populations is higher. Estimation of the number of carriers of karyotypic abnormalities in a sample from a population of young horses of various breeds, using molecular cytogenetic techniques. Cross-sectional. Venous blood samples were collected from 500 young horses representing 5 breeds (Purebred Arabian, Hucul, Polish primitive horse [Konik], Małopolska, Coldblood, Silesian). Chromosomes and DNA were obtained from blood lymphocytes and evaluated by fluorescence in situ hybridisation (FISH) and PCR, using probes and markers for the sex chromosomes and select autosomes. Nineteen horses, 18 mares and 1 stallion, were diagnosed with different chromosomal abnormalities: 17 cases of mosaic forms of sex chromosome aneuploidies with a very low incidence (0.6%-4.7%), one case of a SRY-negative 64,XY sex reversal mare, and one mare with X-autosome translocation. The percentage of sex chromosomal aberrations was established as 3.8% in the whole population, 6.08% in females and 0.49% in males. Limited sample size, confined to horses from Poland. The rate of sex chromosomal abnormalities we identified was almost double that reported in previous population studies that used classical chromosome staining techniques. FISH allowed the detection of aneuploid cell lines which had a very low incidence. The FISH technique is a faster and more precise method for karyotype examination; however, it is usually focused on only one or two chromosomes while banding karyotyping includes the entire chromosome set.

External Validation of PRISM Score in Multiple Myeloma Patients in a Community Setting: A Retrospective Cohort Study

Background: Newly diagnosed Multiple Myeloma (MM) patients have a 10 to 20 times higher risk of developing venous thromboembolism (VTE) than the general population. The pathogenesis may involve several factors such as activation of procoagulant factors, acquired activated protein C resistance, and inflammation. In addition to the general risk factors for venous thromboembolism such as older age, immobility, and surgery, some MM treatment-related factors such as the use of immunomodulatory drug therapy and abnormal cytogenetics contribute to the increased risk. The PRISM score (Prior VTE, Race, IMiD, Surgery, Metaphase Cytogenetics) is a risk prediction tool for VTE in MM and was published in 2022. The score ranges from 0 to 18 and patients were stratified into three groups: low (0), intermediate (1-6), and high risk (≥ 7). Our primary objective was to assess the external validity of the PRISM score in our community hospital setting. Methods: All consecutive newly diagnosed MM treated at Saint Vincent Hospital from 1/1/2008 to 12/22/2022 were included in our analysis. Patients receiving therapeutic anticoagulation for other indications or a diagnosis of VTE within 6 months preceding the MM diagnosis were excluded. Variables were collected through a retrospective chart review during MM treatment initiation. Model discrimination was assessed by calculating the area under the receiver operating characteristic curve (AUROC). Logistic regression was used to calculate the odds ratio (OR) of VTE occurrence in the risk prediction model. Results: A total of 214 patients with newly diagnosed MM and available data on VTE occurrence were included. The median age of our cohort at treatment initiation was 74 years (range 31- 96). The median BMI was 27.8. 56% were male, and 44% were female. The mean PRISM score was 2.9, and 73% of MM were IgG/IgA subtype. IMiD-based induction regimen was used in 63 % of patients. A total of 24 patients (11.2%) were diagnosed with VTE after treatment initiation, of which 8 (3.7%) patients had a previous history of VTE. Prophylactic aspirin was used in 53.8% and abnormal metaphase cytogenetics was identified in 13% of MM cases. The median time to VTE from induction therapy was 4.8 months. The PRISM score was low in 116 patients, intermediate in 89, and high in 9 patients. The AUROC of the model was 0.759 (95% CI, 0.644-0.874) (1). The cumulative incidence of VTE at 6 months, 12 months, and more than 1 year was 6%, 7%, and 11.2% of the patients respectively. Each 1-point increase in the score was associated with a significantly higher chance of VTE occurrence in the model [ HR= 1.53, 95% CI (1.27-1.85), P<0.001]. Among the parameters, patients with a history of VTE [ HR= 1.55, 95% CI (1.26-1.91), P<0.001] and with abnormal metaphase cytogenetics [HR= 2.35, 95% CI (1.37-4.05), P<0.001] had a higher strength of association with the development of VTE after induction (2). Furthermore, among 9 patients with a high-risk PRISM score (≥ 7), 7 (77%) developed VTE within the first 6 months after treatment initiation. Conclusions: Our findings suggest that PRISM [AUROC=0.75, 95% CI (0.644-0.874), P<0.001] score could statistically predict VTE outcomes in our patient population. Abnormal metaphase cytogenetics was a significant predictor of VTE in the validation cohort. Abnormal cytogenetics has been well established to be an indicator of aggressive tumor biology and proliferation in stroma-independent MM. It has been established to be a predictor of VTE in acute myeloid leukemia as well. Also, high-risk scores were predictive of earlier development of VTE after treatment induction. Our study strengthens the evidence of using PRISM as a predictive tool. Further studies are needed to enhance the sensitivity of our existing tool.

Production of sterile mono-sex triploid yellow drum (Nibea albiflora): genotypic females and sex-reversed phenotypic males with emphasis on utilization as surrogate broodstock.

Production of sterile mono-sex fish is of great significance for sustainable aquaculture as well as germ cell transplantation. In this study, we aimed to produce mono-sex triploid yellow drum, including genotypic females (XXX female) and sex-reversed phenotypic males (XXX male). Firstly, the mono-female triploids were produced through cold-shock treatment on eggs fertilized with sperm from neo-males. Then, the mono-male triploids were produced by the sex reversal of mono-female triploids with oral administration of letrozole (LZ). We comparatively investigated the growth and gonadal development in the mono-sex triploids. The results showed that the triploids displayed similar growth performance to their diploids throughout their first year, but had impaired gonadosomatic index and gametogenesis. No mature gametes were produced in the triploids during their first spawning season. Meanwhile, we analyzed the process of gametogenesis in the both sex of triploids. Ultrastructure of gametogenesis showed that the germ cells arrested at abnormal metaphase 1 in females, while males had irregular meiotic divisions, variable-sized spermatid and degenerated cells. The expression levels of meiosis-related genes (i.e., sycp3 and rec8) confirmed the abnormal meiosis in the triploids. Furthermore, the gonadal development was also determined by the expression patterns of vasa, dmrt1 and cyp19a1a. Abnormal expression of vasa mRNA and protein were detected in triploids. High cyp19a1a expression levels suggested the sex steroid hormones production might be at least partially functional in triploid females. In addition, high dmrt1 expression levels confirmed the masculinization and testicular development of sex-reversed triploid males by LZ. Our findings provide an efficient protocol to produce sterile mono-sex triploid yellow drum and provide new insights into the mechanism of gonadal sterility of triploid fish.

Cytogenomic Profiling of Chronic Lymphocytic Leukaemia Patients Using DNA Microarray

Introduction: Chronic lymphocytic leukaemia (CLL) is the most frequent adult leukaemia in the Western world. The clinical presentation varies greatly, from very indolent cases to those with aggressive and fast advancing disease. This variation has significant implications for clinical approaches, therapeutic tactics, and, ultimately, survival durations from diagnosis. Acquired chromosomal aberrations play a key role in CLL aetiology. Due to difficulty to obtain abnormal metaphases for analysis, few methods such as fluorescence in-situ hybridization (FISH) and multiplex ligation-dependent probe assay (MLPA) were employed to detect chromosomal aberration however the methods are limited to specific locus only. Thus, this study is aimed to detect the chromosomal aberrations using DNA microarray platform. Methods: In this retrospective study, DNA archive obtained from 7 CLL patients which collected at diagnosis and subjected to Affymetrix CytoScan® 750K single nucleotide polymorphism (SNP) array following the manufacture procedure. The raw data obtained were analysed using the Chromosome Analysis Suite (ChAS) software (Affymetrix) using annotations of genome version GRCh38 (hg38). Result: Out of 7 patients, 4 of them showing deletion of 13q while 3 of them showing deletion of 14q in various region . Some of the deleted loci were too small (0.42-0.6Mb) to be detected by conventional cytogenetic analysis (CCA). There was also the presence of additional chromosomal aberrations that could be missed by CCA, FISH, or MLPA due to cryptic deletion or duplication that was as small as 0.4MB in size. Conclusion: The present study showed that low resolution chromosomal aberration was able to be detected using DNA microarray platform in comparison to CCA, FISH and MLPA.

Spectrum of stable and unstable rearrangements in lymphocytic chromosomes investigated in Bhopal population 30 years post MIC disaster amid co-exposure to lifestyle, living, and occupational hazards.

Immediate assessment of genetic damage in methyl isocyanate (MIC) gas-exposed population in small and heterogeneous samples using diversified study designs and solid-stained metaphases could not depict the actual genetic impact of MIC on accidentally exposed individuals. The outcome of the then large multi-center genetic screening program was not available to the public and scientific community. Also, the routine and regular epidemiological health survey does not capture the genetic and long-term effect of MIC. Therefore, genetic screening was carried out 30 years post disaster during 2015-2017 with a view to screen the present status of chromosomal consequences in lymphocytic cells. Participants were recruited from moderate (34) and severely (78) exposed and unexposed (35) cohorts with their informed consent. Analysis of ~100 mitotic cells and karyotyping of at least 10-15 and all abnormal metaphases detected structural and numerical alterations, including stable and replicable ones. Clonal abnormalities were detected with monosomal and complex karyotypes, trisomy 8, del5q/20q, loss of Y, etc. Among all, X-chromosome was frequently involved in numerical alterations. Structural aberrations appeared higher in the then exposed populations, though abnormalities cannot be linked directly to MIC exposure 30 years post disaster. Collectively, all rearrangements were markedly higher in the severely exposed population. Altogether, the detected abnormalities appeared random and indicated genomic instability, suggesting follow-up at shorter intervals for the individuals detected with clonal aberrations. G-banding has facilitated recognition of chromosomal involvement and their breakpoints and classification of structural rearrangements. The present data has been derived from the 30-year post-disaster genetic screening.

Abnormal metaphase cytogenetics predicts venous thromboembolism in myeloma: derivation and validation of the PRISM score

AbstractAlthough venous thromboembolism (VTE) is an important treatment and disease-related complication in myeloma, a validated risk prediction model including disease-specific variables such as cytogenetics or tumor burden is lacking. The aim of this study was to develop a new risk prediction model for VTE in the context of modern antimyeloma therapy. All consecutive patients diagnosed at the Cleveland Clinic between 2008 and 2018 and with available data on baseline candidate risk factors constituted the derivation cohort. The primary outcome was VTE (deep venous thrombosis/pulmonary embolism) within 1 year of treatment initiation. A multivariable model was used, and weights were derived from subdistribution hazard ratios to construct a risk score. The model was validated both by internal bootstrap validation and in an external validation cohort. The derivation cohort consisted of 783 patients. A 5-component risk prediction tool, named the PRISM score, was developed, including the following variables: prior VTE, prior surgery, immunomodulatory drug use, abnormal metaphase cytogenetics, and Black race. The c-statistic of the model was 0.622 (95% confidence interval [CI], 0.567-0.674). The model stratified patients into low, intermediate, and high risk, with 12-month cumulative VTE incidence of 2.7%, 10.8%, and 36.5%, respectively. Risk of VTE increased significantly with increasing score in both the derivation and the external validation data sets, with a subdistribution hazard ratio per 1-point increase of 1.28 (95% CI, 1.19-1.39; P < .001) and 1.23 (95% CI, 1.07-1.41; P = .004) respectively. Although the PRISM score can guide clinicians in identifying patients at a high risk of VTE, additional external validation is necessary for incorporation into routine clinical practice.

Diet-induced maternal obesity and overnutrition cause a decrease in the sperm quality of the offspring

The present work aimed to study the changes caused by maternal obesity and overnutrition in both the quality and function of spermatozoa of the offspring. To this end, female rats received either a standard or cafeteria diet from 22 days of age until the weaning of their offspring, and the male offspring from rats fed the standard and cafeteria diet (OSD and OCD respectively) were used. Different endpoints in the offspring, as body weight, weight gain, and glycemia were recorded and the testes were removed at 60 days of age. Different spermatozoa parameters, such as mitochondrial function, functional integrity of the sperm plasma membrane, capacitation, and acrosome status, were evaluated. The OCD group was heavier than the OSD group and exhibited lower testis and epididymal indices. The OCD group also showed a decrease in the ability of the sperm tail to react in the presence of a hypoosmotic solution, deficiency in sperm mitochondrial function, a lower percentage of spermatozoa without acrosome when exposed to a capacitation medium, and a higher number of abnormal metaphases. In addition, compared with OSD, OCD rats had a higher number of TUNEL-positive cells in the histological sections of the testis, and greater presence of reactive oxygen species in the spermatozoa, evaluated by a fluorescent probe. However, the OCD group displayed lower protein levels of cytochrome c and caspase-3 in testis tissue than the control group. These results suggest that maternal obesity and overnutrition program the offspring to develop poor sperm quality and function, which may imply a condition of subfertility.

Evaluation of cytotoxicity and genotoxicity effects of refractory pollutants of untreated and biomethanated distillery effluent using Allium cepa

Environmental pollution caused by the discharge of raw and partly treated distillery effluent has become a serious and threatening problem due to its high pollution load. The aim of the present study was to assess the physicochemical load in alcohol distillery effluent before and after biomethanation treatment and the cyto- and genotoxicity effects of refractory pollutants emanated in raw/untreated and biomethanated distillery effluent on the ultrastructural and biochemical responses of Allium cepa root tip cells. Physicochemical analysis revealed high biochemical oxygen demand (BOD: 47840-36651 mg L−1), chemical oxygen demand (COD: 93452-84500 mg L−1) and total dissolved solids (TDS: 64251–74652 mg L−1) in raw and biomethanated effluent along with metal(loid)s (Fe: 456.152–346.26; Zn: 1.654–1.465; Cu: 0.648–0.562; Ni: 1.012–0.951, and Pb: 0.264 mg L−1) which were beyond the safe discharge values prescribed by the environmental regulatory agencies. The UV–Visible and Fourier transform infrared spectrophotometry analyses confirmed the high levels of organic, inorganic, and mixed contaminants discharged in raw and biomethanated distillery effluents. Furthermore, GC–MS analysis characterised chemical contaminants, such as hexadecanoic acid, butanedioic acid, bis(trimethylsilyl) ester; hexadecane, 2,6,11,15-tetramethyl, stigmasterol, and β-sitosterol trimethylsilyl ether that have been reported as androgenic-mutagenic, and endocrine disrupting chemicals by the United States Environmental Protection Agency (U.S. EPA). The cytotoxicity measured by A. cepa showed dose depended inhibition root growth inhibition and simultaneous reduction in mitotic index in tested effluents. The chromosomal aberrations studies resulted in laggard chromosomes, sticky chromosomes, vagrant chromosomes, chromosome loss, c-mitosis, chromosome bridge, abnormal metaphase, and disturbed anaphase as found in a dose-dependent manner. Furthermore, dose-dependent enhancement in the levels of malondialdehyde, hydrogen peroxide, and antioxidative enzymes, such as superoxide dismutase, ascorbate peroxidase, and catalase were found to be higher in raw effluents treated root cells compared to biomethanated distillery effluent. Analysis of ultrastructural changes in root tip cells by TEM analysis revealed dramatic changes in the morphology of cell organelles and accumulation of metallic elements in and on the surface tissues. The results concluded that the discharged distillery effluents retained certain toxic pollutants which imposed cytotoxic and genotoxic hazards to A. cepa. Thus, for the sake of environmental protection, the raw as well as the disposed biomethanated effluent must be efficiently treated before its dumping into the terrestrial ecosystem.

Assessment of Cytogenotoxicity Induced by Lead Acetate on Root Tip Cells of Onion (Allium cepa L.)

The aim of present study was to assess the cytotoxic and genotoxic potency of lead acetate using an Allium root tip bioassay. Root tips of Allium cepa L. were treated with different concentrations of lead acetate viz., 0.03, 0.06, and 0.09 mg mL-1 of lead acetate for 48 h. Mitotic index (MI), phase index (PI), and total chromosomal abnormalities (TA) were calculated. The effect of lead acetate was found to be dose-dependent. The frequency of MI ranged from 8.4 to 5.7%, PI of prophase from 4.2 to 3.4%, PI of metaphase from 1.14 to 1.11%, PI of anaphase from 1.8 to 0.6%, and PI of telophase from 1.1 to 0.4% The total chromosomal abnormality (TA) ranged from 0.42 to 1.39% The differences between the averages of all the studied properties were significantly affected by the heavy metal lead acetate concentration variant applied. Mitotic observation showed that increasing of concentration variants doses decrease mitotic index and increased chromosomal abnormality percentage. Various types of physiological and clastogenic (laggard, stickiness, abnormal metaphase, chromatin bridge, and delayed anaphase were observed in Allium cepa root tip cells. Moreover, the A. cepa root tip bioassay method gives critical data for evaluating an agent's action mechanisms and impacts on genetic material.

Clinical implications of loss of bone marrow minimal residual disease negativity in multiple myeloma

AbstractMultiple myeloma (MM) patients frequently attain a bone marrow (BM) minimal residual disease (MRD) negativity status in response to treatment. We identified 568 patients who achieved BM MRD negativity following autologous stem cell transplantation (ASCT) and maintenance combination therapy with an immunomodulatory agent and a proteasome inhibitor. BM MRD was evaluated by next-generation flow cytometry (sensitivity of 10−5 cells) at 3- to 6-month intervals. With a median follow-up of 9.9 years from diagnosis (range, 0.4-30.9), 61% of patients maintained MRD negativity, whereas 39% experienced MRD conversion at a median of 6.3 years (range, 1.4-25). The highest risk of MRD conversion occurred within the first 5 years after treatment and was observed more often in patients with abnormal metaphase cytogenetic abnormalities (95% vs 84%; P = .001). MRD conversion was associated with a high risk of relapse and preceded it by a median of 1.0 years (range, 0-4.9). However, 27% of MRD conversion-positive patients had not yet experienced a clinical relapse, with a median follow-up of 9.3 years (range, 2.2-21.2). Landmark analyses using time from ASCT revealed patients with MRD conversion during the first 3 years had an inferior overall and progression-free survival compared with patients with sustained MRD negativity. MRD conversion correctly predicted relapse in 70%, demonstrating the utility of serial BM MRD assessment to complement standard laboratory and imaging to make informed salvage therapy decisions.

Real World Validation of VTE Risk Models in Newly Diagnosed Multiple Myeloma in a Community Setting

Background:Multiple Myeloma (MM) is associated with increased risk for venous thromboembolism (VTE). Treatment, such as dexamethasone, immunomodulatory drugs (IMID), alkylating agents, and doxorubicin, alter hemostatic pathways and thus promote thrombogenesis 1. MM patients with VTE have a 3-fold increase in mortality compared to those without VTE, so identifying those at risk and aiming to prevent VTE events is important 2. Several clinical VTE risk prediction scores have been developed, including the SAVED score, IMPEDE VTE score, and more recently the PRISM score 2,4,5. The National Comprehensive Cancer Network suggests that patient with MM on IMID therapy should be on aspirin, or therapeutic anticoagulation for those at “high risk”3. However, it remains unclear which risk model, if any, should be used.Our objective was to validate the three published risk assessment tools in a community setting and assess the predictive ability of each.Methods:We conducted a retrospective chart review of all patients with newly diagnosed multiple myeloma who started chemotherapy at Gundersen Health System (La Crosse, WI) between 2010 and 2020 who had at least 6 months of follow up documented. Patients with prior indication for ongoing therapeutic anticoagulation or a diagnosis of VTE within 6 months prior to starting therapy were excluded. Total scores for IMPEDE VTE, SAVED and PRISM scores were calculated from the chemotherapy start date. Statistical analysis included Chi-square, Fisher's exact and Wilcoxon rank sum tests, and Kaplan Meier survival analysis. A p-value ≤ 0.05 was considered significant and all analysis was completed in SAS version 9.4.Results:Our cohort contained 123 patients diagnosed with MM. Average age was 68 years (SD 12.1, range 37-92). Our study included 68 (55%) males and 55 (45%) females with 121 (98%) being White/Caucasian. The mean BMI of patients was 29.4 kg/m2 (SD 7.0, range 18.6-54.4). Kaplan Meier survival analysis showed a 5-year survival rate of 53.1% (95% CI [42.7%, 63.4%]). In the entire cohort, 10 (8.1%) patients were diagnosed with VTE (as compared to 5.8% in IMPEDE study, 8.7% in SAVED study and 8.2% in PRISM study) with 80.0% occurring within 6 months of treatment start date. Aspirin was the most frequently used agent for thromboprophylaxis with 88 (86.3%) patients receiving either 81, 162, or 325 mg of aspirin. IMID therapy was given to 76 (61.8%) patients, 114 (92.7%) received dexamethasone and 114 (92.7%) received proteasome inhibitors. Amongst those on IMIDs, 72 (94.7%) patients received prophylaxis, most commonly aspirin. Abnormal metaphase cytogenetics were noted in 104 (85.4%) patients.Neither the IMPEDE VTE (p=0.6), SAVED (p=0.9) nor PRISM risk scores (p=0.3) were able to statistically predict VTE outcome in our patient population. Using the IMPEDE score, 7 patients in the intermediate risk group and 3 patients in the low-risk group had a VTE. In the SAVED model, 5 patients in the low-risk group and 5 patients in the high-risk group had a VTE. Using the PRISM risk score, all 10 of the patients with VTE were in the intermediate risk group. Most patients who were on IMID therapy fell into the intermediate risk group on the IMPEDE VTE and PRISM scoring systems, and the SAVED score had an approximately equal patient distribution between the high risk and low risk group.Conclusions:Our patients with multiple myeloma had similar rates of VTE as compared to the published models, with the majority occurring in the first 6 months of chemotherapy. In total, our patients on IMID therapy received appropriate prophylaxis with aspirin. Overall, 8.1% of our patients had a VTE event. However, none of the three risk models were able to predict the development of VTE. In fact, many of the VTE events occurred in patients who were felt to be low or intermediate risk. While the sample size is small and from a single health system, we had excellent follow up and ability to closely examine each chart for treatment and outcomes. Further efforts should focus on collaboration across institutions to increase the sample size, to validate and compare existing models. The majority of myeloma treatment occurs in the community; thus, it is important to ensure the findings are reproducible in that patient population. DisclosuresNo relevant conflicts of interest to declare.

Impact of cytogenetic abnormalities on outcomes of adult Philadelphia-negative acute lymphoblastic leukemia after allogeneic hematopoietic stem cell transplantation: a study by the Acute Leukemia Working Committee of the Center for International Blood and Marrow Transplant Research

Cytogenetic risk stratification at diagnosis has long been one of the most useful tools to assess prognosis in acute lymphoblastic leukemia (ALL). To examine the prognostic impact of cytogenetic abnormalities on outcomes after allogeneic hematopoietic cell transplantation, we studied 1731 adults with Philadelphia-negative ALL in complete remission who underwent myeloablative or reduced intensity/non-myeloablative conditioning transplant from unrelated or matched sibling donors reported to the Center for International Blood and Marrow Transplant Research. A total of 632 patients had abnormal conventional metaphase cytogenetics. The leukemia-free survival and overall survival rates at 5 years after transplantation in patients with abnormal cytogenetics were 40% and 42%, respectively, which were similar to those in patients with a normal karyotype. Of the previously established cytogenetic risk classifications, modified Medical Research Council-Eastern Cooperative Oncology Group score was the only independent prognosticator of leukemia-free survival (P=0.03). In the multivariable analysis, monosomy 7 predicted post-transplant relapse [hazard ratio (HR)=2.11; 95% confidence interval (95% CI): 1.04-4.27] and treatment failure (HR=1.97; 95% CI: 1.20-3.24). Complex karyotype was prognostic for relapse (HR=1.69; 95% CI: 1.06-2.69), whereas t(8;14) predicted treatment failure (HR=2.85; 95% CI: 1.35-6.02) and overall mortality (HR=3.03; 95% CI: 1.44-6.41). This large study suggested a novel transplant-specific cytogenetic scheme with adverse [monosomy 7, complex karyotype, del(7q), t(8;14), t(11;19), del(11q), tetraploidy/near triploidy], intermediate (normal karyotype and all other abnormalities), and favorable (high hyperdiploidy) risks to prognosticate leukemia-free survival (P=0.02). Although some previously established high-risk Philadelphia-negative cytogenetic abnormalities in ALL can be overcome by transplantation, monosomy 7, complex karyotype, and t(8;14) continue to pose significant risks and yield inferior outcomes.

Perfluorononanoic acid impedes mouse oocyte maturation by inducing mitochondrial dysfunction and oxidative stress

Perfluorononanoic acid (PFNA), a member of PFAS, is frequently detected in human blood and tissues, even in follicular fluid of women. The exposure of PFNA, but not PFOA and PFOS, is positively correlated with miscarriage and increased time to pregnancy. Toxicological studies indicated that PFNA exposure is associated with immunotoxicity, hepatotoxicity, developmental toxicity, and reproductive toxicity in animals. However, there is little information regarding the toxic effects of PFNA on oocyte maturation. In this study, we investigated the toxic effects of PFNA exposure on mouse oocyte maturation in vitro. Our results showed that 600 μM PFNA significantly inhibited germinal vesicle breakdown (GVBD) and polar body extrusion (PBE) in mouse oocytes. Our further study revealed that PFNA induced abnormal metaphase I (MI) spindle assembly, evidenced by malformed spindles and mislocalization of p-ERK1/2 in PFNA-treated oocytes. We also found that PFNA induced abnormal mitochondrial distribution and increased mitochondrial membrane potential. Consequently, PFNA increased reactive oxygen species (ROS) levels, leading to oxidative stress, DNA damage, and eventually early-stage apoptosis in oocytes. In addition, after 14 h culture, PFNA disrupted the formation of metaphase II (MII) spindle in most PFNA-treated oocytes with polar bodies. Collectively, our results indicate that PFNA interferes with oocyte maturation in vitro via disrupting spindle assembly, damaging mitochondrial functions, and inducing oxidative stress, DNA damage, and early-stage apoptosis.

Abnormal meiosis in fertile and sterile triploid cyprinid fish.

Meiosis is the key process for producing mature gametes. A natural fertile triploid Carassius auratus population (3nDTCC) and an artificially derived sterile triploid crucian carp (3nCC) have been previously observed, providing suitable model organisms for investigating meiosis characteristics in triploid fish. In the present study, the microstructures and ultrastructures of spermatogenesis were studied in these fishes. TdT-mediated dUTP nick end labeling detection was performed to investigate the apoptosis of spermatocytes. Fluorescence in situ hybridization was employed to trace chromatin pairing. In addition, the mRNA expressions of cell cycle-related genes (i.e., cell division control 2 and cell cycle protein B) were determined by quantitative realtime polymerase chain reaction to illustrate the molecular mechanism of abnormal meiosis in the 3nCC. The results showed that the 3nCC undergoes an irregular prophase I, with the chromosomes distributed in a unipolar radial manner and exhibiting partial pairing, hindered metaphase I, and degenerated cells in the subsequent stages. Meanwhile, the 3nDTCC presented a relatively regular meiotic prophase I with complete conjugate chromosome pairs and chromosomes distributed along the karyotheca, which were presented as a ring structure by slicing. Only the spreads with 130-150 irregular chromosomes can be easily detected in the 3nDTCC, suggesting that it may undergo an abnormal metaphase I. This study provides new insights into the meiosis of fertile and sterile triploid cyprinid fish.

Abnormal Metaphase Cytogenetics Adds to Currently Known Risk-Factors for Venous Thromboembolism in Multiple Myeloma: Derivation of the PRISM score

Background: Prevention and management of venous thromboembolic events [VTE] is an important component of supportive care in newly diagnosed multiple myeloma [MM], especially in the era of immunomodulatory drugs [IMiDs]. Recently, two validated risk assessment models [RAMs], SAVED and IMPEDE-VTE, were developed to identify patients at high risk of VTE. However, these models have following limitations: (1) Patients were not uniformly treated in the era of contemporary MM therapy (2) Disease-specific variables were not available in the databases from which these scores were derived. Our primary aim was to develop a simple predictive model for VTE in MM using patient-specific, disease-specific, and treatment-specific variables. Our secondary aim was to assess the impact of VTE on overall survival [OS]. Methods: All consecutive patients with newly diagnosed MM treated at Cleveland Clinic from 1/1/2008 to 12/31/2018 were included in our analysis. The primary objective was to identify baseline variables associated with VTE within 12 months of treatment initiation. Candidate variables included those in IMWG, SAVED, and IMPEDE-VTE models as well as additional risk-factors from literature review in MM and cancer-associated VTE. Stepwise selection with variable entry criterion of p&amp;lt;0.20 and a variable retention criterion of p&amp;lt;0.05 was used to identify significant factors on multivariable analysis [MVA]. RAM was developed by subtracting 1 from the hazard ratio of a potential variable, rounding to the nearest 0.5, and multiplying by 2 to obtain a whole number. The impact of VTE on OS was assessed with landmark analysis. Results: A total of 934 patients with newly diagnosed MM and available data on VTE occurrence were considered for inclusion. We excluded patients with VTE within 6 months before starting therapy [n=5] and patients on therapeutic anticoagulation or receiving &amp;gt;1 prophylactic regimen [n=146], resulting in a total of 783 patients for model development. The most common induction regimen was bortezomib [V]-lenalidomide [R]-dexamethasone [VRD; 41%], followed by VD [22%], RD [20%], V-cyclophosphamide-dexamethasone [VCD; 11%], and others [7%]. Median age at treatment initiation was 63 years [range, 22-91], 55% were males, and 20% were Blacks. ISS stage III disease was present in 32%, high-risk FISH in 23%, abnormal metaphase cytogenetics in 18%, and serum creatinine &amp;gt;2 mg/dl in 19% of patients. Notably, 76% had received a dexamethasone dose of 120-160 mg/cycle, with only 5.9% started on a higher dose [&amp;gt;160 mg/cycle]. The most common thromboprophylaxis agent was aspirin [60%], followed by low molecular weight heparin [LMWH; 3.8%]; 37% of patients received no thromboprophylaxis. Erythropoietin and intravenous immunoglobulin were used in 2.9% and 1.2% of patients respectively. Median time to VTE from treatment initiation was 3.2 months. Cumulative incidence of VTE at 6 and 12 months was 8.2% [95% CI, 6.6-10.1] and 11.5% [95% CI, 9.5-13.6] respectively. Factors significantly associated with development of VTE on MVA were combined to develop the PRISM score [Table 1]: Prior VTE history [HR 5.06; 8 points], Black Race [HR 1.71; 1 point], IMiD use [HR 2.17; 2 points], Surgery within 3 months [HR 3.44; 5 points], and abnormal Metaphase cytogenetics [HR 2.10; 2 points]. The theoretical score range is 0-18, with a HR of 1.28 per 1-point increase in score [c-statistic 0.62]. Internal bootstrap validation including 1,000 samples showed a c-statistic of 0.62 [IQR, 0.60-0.64]. Using three risk groups by recursive partitioning analysis, 17.8%, 74%, and 8.1% belonged to low [0], intermediate [1-6], and high-risk [&amp;gt;6] groups respectively. The 12-month cumulative incidence of VTE in the 3 respective groups were 2.7%, 10.8%, and 36.5% [Figure 1]. Occurrence of VTE in the first 12 months was not associated with worse OS on landmark analysis at 3, 6, 9, and 12 months. Conclusion: We have developed and internally validated a RAM for VTE in MM in the context of contemporary MM therapy including disease-specific variables. Studies of external validation and comparison with existing RAMs are warranted. The PRISM Score could be used to identify high-risk patients for thromboprophylaxis. Figure Disclosures Valent: Amgen Inc.: Other: Teaching, Speakers Bureau; Takeda Pharmaceuticals: Other: Teaching, Speakers Bureau; Celgene: Other: Teaching, Speakers Bureau. Khouri:Sanofi Genzyme: Other: Advisory Board. Anwer:Incyte, Seattle Genetics, Acetylon Pharmaceuticals, AbbVie Pharma, Astellas Pharma, Celegene, Millennium Pharmaceuticals.: Honoraria, Research Funding, Speakers Bureau. Khorana:Pharmacyclics: Honoraria; Pfizer: Honoraria; Sanofi: Honoraria; Medscape: Honoraria; Leo Pharma: Honoraria; Seattle Genetics: Honoraria; Pharmacyte: Honoraria; Leap: Research Funding; Bayer: Honoraria; Janssen: Honoraria; Merck: Research Funding; Array: Other: Research funding (to institution); BMS: Honoraria, Research Funding.

Late Relapsing Multiple Myeloma ≥ 10 Years after Treatment on Total Therapy Protocols Are Associated with Good Outcome

Introduction Novel agents are incorporated into a backbone of multi-agent chemotherapy and tandem transplantation in successive Total Therapy (TT) protocols for multiple myeloma (MM) patients. The TT protocols extend the duration of remission with a significant proportion of patients achieving long-term disease control. However, a small percentage of patients have documented late relapse and we theorize that these relapses are indolent with overall good clinical outcome. While even a late relapse is seemingly an undesirable outcome, defining the clinical implications is imperative to understanding the impact on long-term prognosis and management. Methods Our MM database was interrogated to identify patients that presented with relapsing disease ≥ 10 years from diagnosis and were treated on TT protocols. All patients had myeloma markers every 2 to 3 months and bone marrows with either PET-CT or MRI at least once a year. In the group of patients with documented relapse, we obtained the pattern of relapse, clinical and laboratory markers, cytogenetics, FISH, gene expression profile (GEP), bone marrow (BM) minimal residual disease (MRD), imaging (PET-CT and MRI, use of salvage transplant, and response assessment including best response to treatment. Patterns of relapses were evaluated based on elevated serum myeloma markers, BM plasmacytosis and presence of macrofocal relapse/extramedullary disease. Clinical relapses were classified based on the International Myeloma Working Group criteria. Indolent relapse was defined as a biochemical relapse with a rising M-protein or free light chains, less than 30% bone marrow plasmacytosis involvement, absence of focal lesion on imaging and of CRAB criteria, low riskGEP70 signature at relapse or no abnormal metaphase cytogenetics. Results A total of 2055 patients were enrolled and treated on successive TT protocols (TT1 to TT7) of which 658 patients had ≥10 years follow up from initial study enrollment. Among these 658 patients, 8% (53/658) had a clinical relapse ≥ 10 years from diagnosis. The median time to clinical relapse was 11. 8 years (range, 9.8 - 17.6). Median age at the time of relapse was 67.7 years with 28% being females. Gene expression profiling showed that 45% (24/53) belonged to the molecular CD2 and LB subgroups. The most common pattern of relapse is with BM plasmacytosis in 49% (26/53) with 36% (19/53) presenting with CRAB clinical features. Focal lesions by MRI and/or CT-PET were present in 35.8% (19/53). Overall, 56.6% (30/53) of patients had indolent relapses; of these 9 and 7 belonged to the CD2 and LB molecular subgroups, respectively. BM MRD assessment by 8-color flow cytometry was available in 17 patients and MRD positivity preceded clinical relapse by a median 17.9 months (range: 0 - 60.8). Forty-one patients required treatment for disease relapse with 11 patients managed expectantly without treatment. While most patients (73.1%, 30/41) received initial treatment with either PI/IMiD/PI IMiD combinations, 8 patients later underwent salvage ASCT. A response of ≥ VGPR was attained in 70% (31/47) of the treated patients with the large majority (87%) achieving a sCR/CR. The overall survival from relapse at 5 years was 73.3% (95% CI: 55.0% to 85.1%). A total of 14/53 patients died during follow-up;7 patients died to progressive disease; 6 of these 7 presented with focal lesions detected on imaging and had an aggressive relapse. Three patients died to treatment related complications, 1 had an unrelated pneumonia, the cause of death was indeterminate in 2 and unknown in 1. Overall, in these 14 patients, median time to death from initial diagnosis was 15.4 years (range: 11.8 - 18.0) and a median time to death from relapse was 3.2 years (range: 0.3 - 6.0). Conclusions Late relapses (&amp;gt;10 years) in MM are a rare event occurring in 8% of long-term survivors and accounting for 2.6% of all patients treated on the TT protocols. Intriguingly, most patients with late relapsing MM belong to the GEP subtype LB and CD2 and accounted for 51% of the indolent relapses. These findings have clinical implications and emphasizes the importance of extended and close follow-up even in patients with prolonged clinical remission. Disclosures van Rhee: Karyopharm: Consultancy; Adaptive Biotech: Consultancy; Takeda: Consultancy; EUSA: Consultancy; CDCN: Consultancy.

In vivo protective effects of Ginkgo biloba L. leaf extract against hydrogen peroxide toxicity: cytogenetic and biochemical evaluation.

In this study, the protective effects of Ginkgo biloba leaf extract (GbE) against toxicity induced by hydrogen peroxide (H2O2) in Swiss albino mice were investigated. Abnormal metaphase number (AMn), mitotic index (MI), micronucleus (MN), and chromosomal abnormalities (CAs) were analyzed for cytogenetic effects. Serum aspartate aminotransferase (AST), alanine aminotransferase (ALT), alkaline phosphatase (ALP), blood urea nitrogen (BUN), creatinine, glutathione (GSH), and malondialdehyde (MDA) levels in liver and kidney organs were investigated as indicators of biochemical toxicity. Six experimental groups were formed as a control and treatment group, each containing six animals. The mice in the control group were given tap water, while the mice in the administration group received two different doses of GbE and H2O2 for 45 consecutive days. It was observed that H2O2 administration caused a significant decrease in MI compared to the control group and caused a significant decrease in the frequency of AMn, MN, and CAs. Chromatid break was the most common type of CAs induced by H2O2, and the other CAs types observed in this study were chromosome break, fragment, dicentric, gap, and ring. It has been determined that GbE treatment decreases the clastogenic effects of H2O2 and reduces the MN and CAs frequency and causes a re-increase in mitotic cell numbers. It was determined that H2O2 administration caused changes in biochemical parameters and resulted in significant increases in serum AST, ALP, ALT, BUN, and creatinine levels. However, the level of MDA, which is an indicator of oxidative damage, increased, and GSH level decreased in liver and kidney tissues. Oxidative damage caused by H2O2 in liver and kidney tissues was improved, and all biochemical parameters tested were found to be ameliorated after GbE treatment. This improvement was dependent on the dose of GbE, and improvement in 150mg/kg bw GbE was found to be more prominent. As a result, the GbE can be used as an antioxidant nutritional supplement to protect against the toxic effects of environmental agents such as H2O2.

Real-World Data on Safety and Efficacy of Venetoclax-Based Regimens in Relapsed/Refractory t[11;14] Multiple Myeloma

Background: Venetoclax is an orally bioavailable inhibitor of the anti-apoptotic protein Bcl-2. In Multiple Myeloma [MM] characterized by t[11;14], it is particularly effective with a single-agent response rate of 40%. However, there have been recent safety concerns due to a higher mortality in the venetoclax-arm of BELLINI trial [Kumar et al. EHA 2019]. At our institution, venetoclax has been used off-label for patients with relapsed/refractory t[11;14] MM and AL amyloidosis. Our objective was to present the efficacy and safety data on venetoclax-based regimens at our institution. Methods: The Cleveland Clinic MM database was queried to identify patients meeting the following inclusion criteria: [a] Relapsed or refractory MM [b] Treated with ≥1 cycle of venetoclax-based regimen outside of a clinical trial. All patients meeting the above criteria through April 2019 were included and followed until June 30, 2019. Clinical data were independently reviewed by two investigators, D.B. and R.C., and conflicts were resolved by consensus. The primary endpoint was overall response rate [ORR], per International Myeloma Working Group criteria. Results: A total of ten patients were included in our analysis. Among them, five were males, all were Caucasian, and five had an ECOG performance status of 2 or 3 at treatment initiation. Four patients had ISS stage III at diagnosis. All had t[11;14] translocation, five had deletion [13q], three had deletion [17p], and three had abnormal metaphase cytogenetics with complex karyotype. Patients received a median of 6 lines of treatment prior to venetoclax [range, 2-19]. Notably, 10/10 were refractory to bortezomib, 4 to carfilzomib, 4 to ixazomib, 9 to lenalidomide, 7 to pomalidomide, 3 to elotuzumab, and 8 to daratumumab. Five patients had received prior autologous stem cell transplantation, among whom, two had relapsed within one year post-transplant. A total of five patients had clinical relapse without extramedullary disease, three had clinical relapse with extramedullary disease, and two had biochemical-only relapse at initiation of venetoclax. The most common regimen used was venetoclax in combination with bortezomib and dexamethasone. Venetoclax was initiated at 400 mg for one week and then titrated to 800 mg depending on tolerability and insurance approval. Among nine patients who were evaluable for response, the ORR was 78% [7 out of 9 patients]. Among these seven responding patients, one achieved a complete response, one achieved a very good partial response, four achieved a partial response, and one achieved a minimal response to therapy. One patient [Patient #10 in Table I] who had cardiac amyloidosis achieved an organ response, with around 45% reduction in NT-pro-BNP. The median follow-up in surviving patients was 5.2 months [range, 3.1-13.5 months]. Six patients were alive at most recent follow-up. The cause of death was relapsed MM in three and septic shock/acute hypoxemic respiratory failure due to invasive aspergillosis [in the setting of disease progression] in one patient. The toxicity profile in individual patients has been summarized in Table I. Non-hematologic toxicities seen in more than one patient included fatigue, weight loss, appetite loss, and gastrointestinal toxicity [nausea, diarrhea, and abdominal cramps]. Four out of ten patients required red blood cell or platelet transfusions for hematologic toxicity. Conclusion: Venetoclax is effective in patients with heavily pre-treated relapsed/refractory t[11;14] MM, with six out of nine response-evaluable patients achieving at least a partial response. Common hematologic toxicities included anemia and thrombocytopenia, with four patients requiring transfusions. Fatigue, nausea, abdominal pain, and weight loss were the major symptomatic toxicities observed in our cohort. One bortezomib and daratumumab-refractory patient with concomitant systemic amyloidosis and MM achieved a cardiac organ response after addition of venetoclax. Further studies in larger cohorts of patients are warranted. Disclosures Valent: Amgen corporation: Speakers Bureau; Takeda pharmaceuticals: Speakers Bureau; Celgene corporation: Speakers Bureau. OffLabel Disclosure: Drug: Venetoclax Purpose: Relapsed/Refractory Multiple Myeloma

Metaphase Cytogenetics for Risk Stratification in Newly Diagnosed Multiple Myeloma

Introduction: Abnormal metaphase cytogenetics are associated with inferior survival in newly diagnosed multiple myeloma (MM). These abnormalities are only detected in one third of cases due to the low proliferative rate of plasma cells. It is unknown if metaphase cytogenetics improve risk stratification when using contemporary prognostic models such as the revised international staging system (R-ISS), which incorporates interphase fluorescence in situ hybridization (FISH). Aims: The aims of this study were to 1) characterize the association between abnormalities on metaphase cytogenetics and overall survival (OS) in newly diagnosed MM treated with novel agents and 2) evaluate whether the addition of metaphase cytogenetics to R-ISS, age, and plasma cell labeling index (PCLI) improves model discrimination with respect to OS. Methods: We analyzed a retrospective cohort of 483 newly diagnosed MM patients treated with proteasome inhibitors (PI) and/or immunomodulators (IMID) who had metaphase cytogenetics performed prior to initiation of therapy. Abnormal metaphase cytogenetics were defined as MM specific abnormalities, while normal metaphase cytogenetics included constitutional cytogenetic variants, age-related Y chromosome loss, and normal metaphase karyotypes. Multivariable adjusted proportional hazards regression models were fit for the association between known prognostic factors and OS. Covariates associated with inferior OS on multivariable analysis included R-ISS stage, age ≥ 70, PCLI ≥ 2, and abnormal metaphase cytogenetics. We devised a risk scoring system weighted by their respective hazard ratios (R-ISS II +1, R-ISS III + 2, age ≥ 70 +2, PCLI ≥ 2 +1, metaphase cytogenetic abnormalities + 1). Low (LR), intermediate (IR), and high risk (HR) groups were established based on risk scores of 0-1, 2-3, and 4-5 in modeling without metaphase cytogenetics, and scores of 0-1, 2-3, and 4-6 in modeling incorporating metaphase cytogenetics, respectively. Survival estimates were calculated using the Kaplan-Meier method. Survival analysis was stratified by LR, IR, and HR groups in models 1) excluding metaphase cytogenetics 2) including metaphase cytogenetics and 3) including metaphase cytogenetics, with IR stratified by presence and absence of metaphase cytogenetic abnormalities. Survival estimates were compared between groups using the log-rank test. Harrell's C was used to compare the predictive power of risk modeling with and without metaphase cytogenetics. Results: Median age at diagnosis was 66 (31-95), 281 patients (58%) were men, median follow up was 5.5 years (0.04-14.4), and median OS was 6.4 years (95% CI 5.7-6.8). Ninety-seven patients (20%) were R-ISS stage I, 318 (66%) stage II, and 68 (14%) stage III. One-hundred and fourteen patients (24%) had high-risk abnormalities by FISH, and 115 (24%) had abnormal metaphase cytogenetics. Three-hundred and thirteen patients (65%) received an IMID, 119 (25%) a PI, 51 (10%) received IMID and PI, and 137 (28%) underwent upfront autologous hematopoietic stem cell transplantation (ASCT). On multivariable analysis, R-ISS (HR 1.59, 95% CI 1.29-1.97, p &lt; 0.001), age ≥ 70 (HR 2.32, 95% CI 1.83-2.93, p &lt; 0.001), PCLI ≥ 2, (HR 1.52, 95% CI 1.16-2.00, p=0.002) and abnormalities on metaphase cytogenetics (HR 1.35, 95% CI 1.05-1.75, p=0.019) were associated with inferior OS. IR and HR groups experienced significantly worse survival compared to LR groups in models excluding (Figure 1A) and including (Figure 1B) the effect of metaphase cytogenetics (p &lt; 0.001 for all comparisons). However, the inclusion of metaphase cytogenetics did not improve discrimination. Likewise, subgroup analysis of IR patients by the presence or absence of metaphase cytogenetic abnormalities did not improve risk stratification (Figure 1C) (p &lt; 0.001). The addition of metaphase cytogenetics to risk modeling with R-ISS stage, age ≥ 70, and PCLI ≥ 2 did not improve prognostic performance when evaluated by Harrell's C (c=0.636 without cytogenetics, c=0.642 with cytogenetics, absolute difference 0.005, 95% CI 0.002-0.012, p=0.142). Conclusions: Abnormalities on metaphase cytogenetics at diagnosis are associated with inferior OS in MM when accounting for the effects of R-ISS, age, and PCLI. However, the addition of metaphase cytogenetics to prognostic modeling incorporating these covariates did not significantly improve risk stratification. Disclosures Lacy: Celgene: Research Funding. Dispenzieri:Akcea: Consultancy; Intellia: Consultancy; Alnylam: Research Funding; Celgene: Research Funding; Janssen: Consultancy; Pfizer: Research Funding; Takeda: Research Funding. Kapoor:Celgene: Honoraria; Sanofi: Consultancy, Research Funding; Janssen: Research Funding; Cellectar: Consultancy; Takeda: Honoraria, Research Funding; Amgen: Research Funding; Glaxo Smith Kline: Research Funding. Leung:Prothena: Membership on an entity's Board of Directors or advisory committees; Takeda: Research Funding; Omeros: Research Funding; Aduro: Membership on an entity's Board of Directors or advisory committees. Kumar:Celgene: Consultancy, Research Funding; Janssen: Consultancy, Research Funding; Takeda: Research Funding.

Cytogenotoxic effects of cypermethrin on root growth: Allium sativum as a model system

Cypermethrin is a pyrethroid pesticide used on agricultural farms in Nigeria to control pests of fruits and vegetables but this is highly toxic to aquatic organisms. To determine toxicity of chemicals, Allium cepa is commonly used as an established bioassay however the bulb is used whole. Allium sativum in contrast is able to be split into cloves of smaller units hence this research aims to validate the potential of Allium sativum as a model plant for genotoxicity assessment. In this study, root growth inhibition test and chromosome aberration assay were used and the effective concentration (EC50) of cypermethrin was determined from the root growth curve. Furthermore, the mitotic activities of the root meristem were assessed using light microscopy. Treatment of root meristem of A. sativum with various concentrations of cypermethrin (0.25, 0.50, 0.75 and 1.0 mg/ml) revealed a reduction in the root length and EC50 of 0.44 mg/ml. Also morphological changes such root wilting, dark spots, tenderness of the clove bases and discoloration of the roots were observed. Cytological studies showed a reduction in mitotic index with increasing cypermethrin concentration. Chromosomal aberrations ranging from abnormal metaphases: c-metaphases, disturbed spindles and vagrant metaphases; to abnormal anaphases: laggard chromosomes, chromosome breaks and multipolarities were also recorded. These aberrations reduced with increased concentration of the pesticide leading to the production of lesser number of dividing cells. These show that cypermethrin is genotoxic to the root meristem and A. sativum is a suitable model plant for detecting pyrethroid genotoxicity in plant.