FISH Abnormalities Research Articles

Hydroxylated benzo[c]phenanthrene metabolites cause osteoblast apoptosis and skeletal abnormalities in fish

To study the toxicity of 3-hydroxybenzo[c]phenanthrene (3-OHBcP), a metabolite of benzo[c]phenanthrene (BcP), first we compared it with its parent compound, BcP, using an in ovo-nanoinjection method in Japanese medaka. Second, we examined the influence of 3-OHBcP on bone metabolism using goldfish. Third, the detailed mechanism of 3-OHBcP on bone metabolism was investigated using zebrafish and goldfish. The LC50s of BcP and 3-OHBcP in Japanese medaka were 5.7 nM and 0.003 nM, respectively, indicating that the metabolite was more than 1900 times as toxic as the parent compound. In addition, nanoinjected 3-OHBcP (0.001 nM) induced skeletal abnormalities. Therefore, fish scales with both osteoblasts and osteoclasts on the calcified bone matrix were examined to investigate the mechanisms of 3-OHBcP toxicity on bone metabolism. We found that scale regeneration in the BcP-injected goldfish was significantly inhibited as compared with that in control goldfish. Furthermore, 3-OHBcP was detected in the bile of BcP-injected goldfish, indicating that 3-OHBcP metabolized from BcP inhibited scale regeneration. Subsequently, the toxicity of BcP and 3-OHBcP to osteoblasts was examined using an in vitro assay with regenerating scales. The osteoblastic activity in the 3-OHBcP (10-10 to 10-7 M)-treated scales was significantly suppressed, while BcP (10-11 to 10-7 M)-treated scales did not affect osteoblastic activity. Osteoclastic activity was unchanged by either BcP or 3-OHBcP treatment at each concentration (10-11 to 10-7 M). The detailed toxicity of 3-OHBcP (10-9 M) in osteoblasts was then examined using gene expression analysis on a global scale with fish scales. Eight genes, including APAF1, CHEK2, and FOS, which are associated with apoptosis, were identified from the upregulated genes. This indicated that 3-OHBcP treatment induced apoptosis in fish scales. In situ detection of cell death by TUNEL methods was supported by gene expression analysis. This study is the first to demonstrate that 3-OHBcP, a metabolite of BcP, has greater toxicity than the parent compound, BcP.

The Cytogenetic Profile of Primary and Secondary Plasma Cell Leukemia: Etiopathogenetic Perspectives, Prognostic Impact and Clinical Relevance to Newly Diagnosed Multiple Myeloma with Differential Circulating Clonal Plasma Cells.

Plasma cell leukemia (PCL) is a rare and aggressive plasma cell dyscrasia that may appear as de-novo leukemia (pPCL) or on the basis of a pre-existing multiple myeloma (MM), called secondary plasma cell leukemia (sPCL). In this prospective study, we have applied a broad panel of FISH probes in 965 newly diagnosed MM (NDMM) and 44 PCL cases of both types to reveal the particular cytogenetic differences among the three plasma cell dyscrasias. In order to evaluate the frequency and patterns of clonal evolution, the same FISH panel was applied both at diagnosis and at the time of first relapse for 81 relapsed MM patients and both at MM diagnosis and during sPCL transformation for the 19 sPCL cases described here. pPCL was characterized by frequent MYC translocations and t(11;14) with a 11q13 breakpoint centered on the MYEOV gene, not commonly seen in MM. sPCL had a higher number of FISH abnormalities and was strongly associated with the presence of del(17p13), either acquired at the initial MM stage or as a newly acquired lesion upon leukemogenesis in the context of the apparent clonal evolution observed in sPCL. In clinical terms, sPCL showed a shorter overall survival than pPCL with either standard or high-risk (t(4;14) and/or t(14;16) and/or del(17p13) and/or ≥3 concomitant aberrations) abnormalities (median 5 months vs. 21 and 11 months respectively, p < 0.001), suggesting a prognostic stratification based on cytogenetic background. These observations proved relevant in the NDMM setting, where higher levels of circulating plasma cells (CPCs) were strongly associated with high-risk cytogenetics (median frequency of CPCs: 0.11% of peripheral blood nucleated cells for high-risk vs. 0.007% for standard-risk NDMM, p < 0.0001). Most importantly, the combined evaluation of CPCs (higher or lower than a cut-off of 0.03%), together with patients’ cytogenetic status, could be used for an improved prognostic stratification of NDMM patients.

Recovery of Haemal Lordosis in European Seabass Dicentrarchus labrax (Linnaeus 1758)

Lordosis of the haemal part of the vertebral column is a frequent abnormality in reared fish. Haemal lordosis develops during the late larval and early juvenile life stages of fish, mainly due to the high swimming activity of the fish in the rearing tanks. In the present study, we have examined whether haemal lordosis recovers during the growth of European seabass. Furthermore, we aimed to develop simple morphometric indices (PrAn1 and PrAn2) that might link the severity of lordosis at the juvenile stage with fish morphological quality at harvesting. At 111 days post-hatching (dph, 53 ± 4 mm standard length, SL), 600 seabass juveniles with lordotic (L, 200 fish) or normal (N, 400 fish) external morphology were selected and introduced in a common tank. At 150 dph (75 ± 7 mm SL), 350 fish were randomly selected, pit-tagged and transferred to a sea cage for on-growing up to 502 dph (234 ± 16 mm SL). The morphological examination of the fish at 150 and 502 dph revealed that 60% (46 out of 77) of L juveniles turned into normal phenotype by the end of on-growing period. Interestingly, 56% of the fish with recovered external morphology (N-Rec) presented either a completely normal vertebral column (31%) or minor abnormalities of individual vertebrae (25%). Following the results of geometric morphometric analysis, the differences in the body shape between N-Rec and N fish were not statistically significant (p &gt; 0.05, canonical variate analysis). The examined morphometric indices were effective in discriminating the normal fish from 58% (PrAn1) to 65% (PrAn2) of lordotic juveniles. Results are discussed with respect to the mechanism of lordosis recovery, and spotlights on their application for quality control and cull out of the abnormal fish in commercial hatcheries.

A simple additive staging system for newly diagnosed multiple myeloma

Risk stratification in multiple myeloma is important for prognostication, patient selection for clinical trials, and comparison of treatment approaches. We developed and validated a staging system that incorporates additional FISH abnormalities not included in the R-ISS and reflects the additive effects of co-occurring high-risk disease features. We first evaluated the prognostic value of predefined cytogenetic and laboratory abnormalities in 2556 Mayo Clinic patients diagnosed between February 2004 and June 2019. We then used data from 1327 patients to develop a risk stratification model and validated this in 502 patients enrolled in the MMRF CoMMpass study. On multivariate analysis, high-risk IgH translocations [risk ratio (RR): 1.7], 1q gain/amplification (RR: 1.4), chromosome17 abnormalities (RR: 1.6), ISS III (RR: 1.7), and elevated LDH (RR: 1.3) were independently associated with decreased overall survival (OS). Among 1327 evaluable patients, OS was 11.0 (95% CI: 9.2–12.6), 7.0 (95% CI: 6.3–9.2), and 4.5 (95% CI: 3.7–5.2) years in patients with 0 (stage I), 1 (stage II), and ≥2 (stage III) high-risk factors, respectively. In the MMRF cohort, median OS was 7.8 (95% CI: NR-NR), 6.0 (95% CI: 5.7-NR), and 4.3 (95% CI: 2.7-NR) years in the 3 groups, respectively (P < 0.001). This 5-factor, 3-tier system is easy to implement in practice and improves upon the current R-ISS.

Real-time detection and tracking of fish abnormal behavior based on improved YOLOV5 and SiamRPN++

In recirculating aquaculture system, the abnormal behavior of fish is usually caused by poor water quality, hypoxia or diseases. Delayed recognition of this behavior will lead to large number of fish deaths. Thus, real-time detection and tracking of fish that behaviors abnormally is an effective way to promote the fish welfare and to improve the survival rate as well as economic benefits of aquaculture. However, due to the high-density breeding, the targets in the fish images are often quite small and in occlusion, which causes high false detection and target loss rate. This article proposes a combined end-to-end neural network to detect and track the abnormal behavior of porphyry seabream. The detection algorithm passes the initial value of the target into the tracking algorithm, and the tracking algorithm tracks subsequent frames to achieve end-to-end abnormal fish behavior detection and achieve high-speed and accurate tracking of abnormal behavior individuals.In the target detection part, YOLOV5s is improved by incorporating multi-level features and adding feature mapping. Compared with the original network, the detection precision AP50:95 is increased by 8.8% while AP50 reaches 99.4%. In the target tracking part, this paper achieves multi-target tracking of abnormal fish based on single-target tracking algorithm SiamRPN++. The tracking precision is 76.7%. By combining the two approaches, individual fish with abnormal behavior can be detected precisely and tracked in real time.

A Phase I/II Study of Twice Weekly Ixazomib Plus Pomalidomide and Dexamethasone in Relapsed and Refractory Multiple Myeloma

A Phase I/II Study of Twice Weekly Ixazomib Plus Pomalidomide and Dexamethasone in Relapsed and Refractory Multiple Myeloma

P-011: 18F-FDG PET/MRI for imaging minimal residual disease evaluation in Multiple Myeloma

<h3>Background</h3> Functional imaging in Multiple Myeloma (MM) plays a crucial role in defining bone disease response. According to IMWG guidelines,18FDG PET/CT represents the only technique recognized to define imaging minimal residual disease (MRD) negativity. More recently, Whole body Diffusion Weighted Imaging (WB DWI)-MRI represents a non ionizing radiation imaging modality with a potential impact on evaluating myeloma bone disease response. Consequently, patients achieving both PET/CT and WB DWI-MRI negativity after treatment showed improved progression free survival (PFS) as compared to patients with single PET or MRI negativity. In the last years, a novel functional technique combining both 18FDG PET with WB DWI-MRI (PET/MRI) has been developed. In this study, we evaluated PET/MRI for bone disease response assessment in MM. <h3>Methods</h3> We retrospectively studied 27 patients (12 female and 15 male) affected by newly diagnosed active MM. All patients received a combined 18F-FDG PET/MRI registering T1w signal, T2w STIR (short tau inversion recovery) signal, DWI (diffusion weighted imaging) signal, mean ADC (apparent diffusion coefficient) value and SUV (standardized uptake volume) max value at diagnosis and at the end of the therapeutic program [after autologous stem cell transplantation (ASCT) or consolidation treatment]. PET and MRI negativity was assessed using Deauville score (DS) and MY-RADS Response Assessment Category (RAC). Median age of the entire cohort was 57 years (44-69). Considering high risk disease features, 7/27 cases were ISS III (25.9%), 2/27 were R-ISS III (7.4%) while 7/26 (26.9%) patients harbored high risk FISH abnormalities [including t(4;14), t(14;16) and del17p]. Bone lytic lesions were detected in almost all patients (24/27, 88.9%), followed by anemia (17/27, 63%), hypercalcemia (3/27, 11.1%) and renal injury (2/27, 7.4%). All patients received bortezomib-thalidomide-dexamethasone induction regimen followed by ASCT. <h3>Results</h3> At the end of the therapeutic program, the overall response rate was 92.6%, with 37% (10/27) achieving a complete response. Regarding baseline PET/MRI evaluation, focal lesions (FLs) were detected in 23 patients, with mean SUVmax of 4.4+/-2.78 and mean ADC of 963+/-264.6 mm2/s. End of treatment PET/MRI showed a PET negativity (DS<3) in 14/27 (51.9%) patients and an MRI negativity (MY-RADS<2) in 18/27 (66.7%) cases. With a median follow up of 33 months, patients with concomitant PET and MRI negativity showed improved PFS with respect to patients with PET or MRI positivity (not reached vs 58 months, p=0.0396). By multivariate analysis, concomitant PET and MRI negativity was associated with better PFS independently from ISS and FISH (p=0.0472). <h3>Conclusions</h3> To our knowledge, this is the first study evaluating PET/MRI for bone disease response in MM. Our data provide evidence that PET/MRI represents a promising tool for imaging MRD negativity evaluation, with patients being both PET and MRI negative showing improved PFS.

P-062: The impact of Bortezomib-based induction on chromosome 1q21 gained newly diagnosed Multiple Myeloma of Chinese origin

<h3>Background</h3> Bortezomib has been reported to favourably impact the outcome of high risk MM predominantly t(4;14) and in some report 17p- its impact on gain 1q21 is unknown. <h3>Methods</h3> To address this deficit, we have analysed CD138-sorted bone marrow plasma cells in cases treated with bortezomib based induction therapy where age, gender, isotype, ISS, LDH and iFISH were available. <h3>Results</h3> 1q+ was identified in 167 (66.8%) of the series and was associated with t(4;14) and high LDH but not with other HR FISH abnormalities. Gain 1q+ was not associated with response rate but did associate with shorter event free survival (EFS) (median EFS: 35 months vs 55 months, p=0.05) and overall survival (OS) (median OS 74 months vs 168, p=0.00025). Thus 1q+ was an independent adverse factors for OS together with ISS3, high LDH, del(17p) and t(4;14), multivariate analysis showed that. Of the cases with 1q+, 75 (44.9%) had 3 copies and 92 (55.1%) had >3 copies of 1q21. Fifty-four (32.3%) had ≥50% cells harbouring 3 copies (gain 1q), and 57 (34.1%) had ≥50% cells with >3 copies. Copy number and clone size did not impact on survival. When a risk score of 1 was assigned to each of 1q+, high LDH, high risk FISH and ISS III, OS was shortened incrementally by a risk score of 0 to 4. Post-relapse/progression survival was inferior in those with 1q+ (median 60 months vs median 118 months, p=0.000316). ASCT improved OS for those with 1q+ (median OS 96 months vs 59 months, p=0.000069). In conclusion, 1q+ is an adverse risk factor for OS in MM irrespective of the use of bortezomib but was partially mitigated by ASCT. <h3>Conclusions</h3> A risk scoring system comprising 1q+, LDH, HR FISH and ISS is a potential tool for risk stratification in MM.

MiR133b Microinjection during Early Development Targets Transcripts of Cardiomyocyte Ion Channels and Induces Oil-like Cardiotoxicity in Zebrafish (Danio rerio) Embryos.

Previous studies have shown that altered expression of a family of small noncoding RNAs (microRNAs, or miRs) regulates the expression of downstream mRNAs and is associated with diseases and developmental disorders. miR133b is highly expressed in mammalian cardiac and skeletal muscle, and aberrant expression is associated with cardiac disorders and electrophysiological changes in cardiomyocytes. Similarly, cardiac dysfunction has been observed in early life-stage mahi-mahi (Coryphaena hippurus) exposed to crude oil, a phenotype that has been associated with an upregulation of miR133b as well as subsequent downregulation of a delayed rectifier potassium channel (IKr) and calcium signaling genes that are important for proper heart development during embryogenesis. To examine the potential role of miR133b in oil-induced early life-stage cardiotoxicity in fish, cleavage-stage zebrafish (Danio rerio) embryos were either (1) microinjected with ∼3 nL of negative control miR (75 μM) or miR133b (75 μM) or (2) exposed to a treatment solution containing 5 μM benzo(a)pyrene (BaP), a model polycyclic aromatic hydrocarbon, as a positive control. At 72 h post fertilization (hpf), miR133b-injected fish exhibited BaP-like cardiovascular malformations, including a significantly increased pericardial area relative to negative control miR-injected embryos, as well as a significantly reduced eye area. qPCR revealed that miR133b microinjection decreased the abundance of cardiac-specific IKr kcnh6 at 5 hpf, which may contribute to action potential elongation in oil-exposed cardiomyocytes. Additionally, ryanodine receptor 2, a crucial calcium receptor in the sarcoplasmic reticulum, was also downregulated by miR133b. These results indicate that an oil-induced increase in miR133b may contribute to cardiac abnormalities in oil-exposed fish by targeting cardiac-specific genes essential for proper heart development.

Issue Highlights-September 2021.

Issue Highlights-September 2021.

Prediction model for the number of crucian carp hypoxia based on the fusion of fish behavior and water environment factors

Fish hypoxia is one of the main tasks of aquaculture monitoring. Abnormal fish hypoxia easily leads to ponding or even mass death of fish, causing serious economic losses to farmers and aquaculture enterprises. In this paper, we propose a model for predicting the number of abnormal crucian carp hypoxia based on the fusion of fish behavior and water environment factors. The cubic convolution interpolation method is used to fuse, synchronize and enhance the calculated number of crucian carp hypoxia with the water environment factors such as dissolved oxygen, pH and temperature. An improved particle swarm optimization (IPSO) algorithm with the dynamically adjusted inertia weight optimizes the initial weights and thresholds of the BP neural network. We adopt an IPSO-BPNN algorithm to construct a non-linear prediction model to predict the number of crucian carp hypoxia. The experimental results show that the model can accurately predict the number within 3 h, and sum of squares due to error (SSE), mean squared error (MSE), mean absolute percentage error (MAPE) of the model are 1.11, 0.015, 0.083, respectively. It shows that the proposed model has good generalization ability and predictive performance.

O-203 Application of machine learning to predict aneuploidy and mosaicism in embryos from in vitro fertilization (IVF) cycles

Abstract Study question Is it possible to predict the likelihood of an IVF embryo being aneuploid and/or mosaic using a machine learning algorithm? Summary answer There are paternal, maternal, embryonic and IVF-cycle factors that are associated with embryonic chromosomal status that can be used as predictors in machine learning models. What is known already The factors associated with embryonic aneuploidy have been extensively studied. Mostly maternal age and to a lesser extent male factor and ovarian stimulation have been related to the occurrence of chromosomal alterations in the embryo. On the other hand, the main factors that may increase the incidence of embryo mosaicism have not yet been established. The models obtained using classical statistical methods to predict embryonic aneuploidy and mosaicism are not of high reliability. As an alternative to traditional methods, different machine and deep learning algorithms are being used to generate predictive models in different areas of medicine, including human reproduction. Study design, size, duration The study design is observational and retrospective. A total of 4654 embryos from 1558 PGT-A cycles were included (January-2017 to December-2020). The trophoectoderm biopsies on D5, D6 or D7 blastocysts were analysed by NGS. Embryos with ≤25% aneuploid cells were considered euploid, between 25-50% were classified as mosaic and aneuploid with &amp;gt;50%. The variables of the PGT-A were recorded in a database from which predictive models of embryonic aneuploidy and mosaicism were developed. Participants/materials, setting, methods The main indications for PGT-A were advanced maternal age, abnormal sperm FISH and recurrent miscarriage or implantation failure. Embryo analysis were performed using Veriseq-NGS (Illumina). The software used to carry out all the analysis was R (RStudio). The library used to implement the different algorithms was caret. In the machine learning models, 22 predictor variables were introduced, which can be classified into 4 categories: maternal, paternal, embryonic and those specific to the IVF cycle. Main results and the role of chance The different couple, embryo and stimulation cycle variables were recorded in a database (22 predictor variables). Two different predictive models were performed, one for aneuploidy and the other for mosaicism. The predictor variable was of multi-class type since it included the segmental and whole chromosome alteration categories. The dataframe were first preprocessed and the different classes to be predicted were balanced. A 80% of the data were used for training the model and 20% were reserved for further testing. The classification algorithms applied include multinomial regression, neural networks, support vector machines, neighborhood-based methods, classification trees, gradient boosting, ensemble methods, Bayesian and discriminant analysis-based methods. The algorithms were optimized by minimizing the Log_Loss that measures accuracy but penalizing misclassifications. The best predictive models were achieved with the XG-Boost and random forest algorithms. The AUC of the predictive model for aneuploidy was 80.8% (Log_Loss 1.028) and for mosaicism 84.1% (Log_Loss: 0.929). The best predictor variables of the models were maternal age, embryo quality, day of biopsy and whether or not the couple had a history of pregnancies with chromosomopathies. The male factor only played a relevant role in the mosaicism model but not in the aneuploidy model. Limitations, reasons for caution Although the predictive models obtained can be very useful to know the probabilities of achieving euploid embryos in an IVF cycle, increasing the sample size and including additional variables could improve the models and thus increase their predictive capacity. Wider implications of the findings Machine learning can be a very useful tool in reproductive medicine since it can allow the determination of factors associated with embryonic aneuploidies and mosaicism in order to establish a predictive model for both. To identify couples at risk of embryo aneuploidy/mosaicism could benefit them of the use of PGT-A. Trial registration number Not Applicable

Chordacentrum mineralization is delayed in zebrafish betaglycan-null mutants.

The Transforming Growth Factor β (TGFβ) family is a group of related proteins that signal through a type I and type II receptors. Betaglycan, also known as the type III receptor (Tgfbr3), is a coreceptor for various ligands of the TGFβ family that participates in heart, liver and kidney development as revealed by the tgfbr3-null mouse, as well as in angiogenesis as revealed by Tgfbr3 downregulation in morphant zebrafish. Here, we present CRISPR/Cas9-derived zebrafish Tgfbr3-null mutants, which exhibited unaltered embryonic angiogenesis and developed into fertile adults. One reproducible phenotype displayed by these Tgfbr3-null mutants is delayed chordacentra mineralization, which nonetheless does not result in vertebral abnormalities in the adult fishes. We also report that the canonical TGFβ signaling pathway is needed for proper chordacentra mineralization and that Tgfbr3 absence decreases this signal in the notochordal cells responsible for this process. Betaglycan's "ligand presentation" function contributes to the optimal TGFβ signaling required for zebrafish chordacentra mineralization.

Incidence and Survival Outcomes Of Extramedullary Myeloma

BackgroundThe survival of patients with multiple myeloma (MM) has considerably improved during the last decade. MM at presentation is mostly limited to the bone marrow, with circulating plasma cells seen in a modest proportion of patients. Organ infiltration or soft tissue involvement by the tumor cells is relatively uncommon at diagnosis; however, several recent reports suggest an increasing number of patients presenting with extramedullary disease at the time of relapse. This could be related to several aspects, especially increased use of more sensitive imaging studies and the newer therapies. We undertook the current study to examine the changes in incidence and potential predisposing causes for development of clinically detected EMD. Patients and MethodsWe retrospectively examined the cohort of 1,051 patients with newly diagnosed MM seen at Mayo Clinic between Jan 2001 and Dec 2010, for their initial evaluation and treatment. Data regarding baseline characteristics, site of EMD, types of therapy and response were extracted from medical records. EMD was defined as a plasmacytoma outside the bone and not contiguous to any bone lesion. Cox proportional hazards model was used to estimate the effect of variables on development of EMD. ResultsThe median age at the diagnosis of MM was 66 years (range, 22-93) with 19% >75 years; 59% were male. The median estimated follow up for the entire cohort was 5.9 years, 492 (47%) were alive at the time of analysis. Over 4139 person years of follow up, 93 patients had an EMD detected (9%), including 32 patients (2.5%) in whom it was found at diagnosis. The median time to detection of EMD among the 61 patients in whom EMD developed after diagnosis, was 26 months (1 mo -10 yrs). The cumulative risk of developing EMD is shown in figure 1A and does not appear to be different between patients seen earlier (2001-2005) vs. (2006-2010), at least during the initial years. The most common sites were skin and soft tissue, lymph nodes, liver/ spleen and pleura/ peritoneum. Twelve patients had a second EMD diagnosed at a median of 5 months from the first EMD. The median OS from diagnosis of MM for the 93 patients with EMD was 3.3 years vs. 5.3 years for the rest; p<0.01. Examining the risk factors associated with development of EMD, an LDH>221 (ULN), PCLI >=3%, bone marrow plasma cell%, presence of high risk FISH abnormalities (Del 17p, t(14;16) and t (14;20) and use of SCT as part of therapy were risk factors for development of EMD. Only, a PCLI >=3 and presence of high-risk FISH increased the risk of developing EMD. [Display omitted] ConclusionExtramedullary disease in multiple myeloma, both present at diagnosis and upon relapse, is associated with poor prognosis. While there is no clear evidence of recent increase in EMD, extended follow-up of patients seen in the recent years may alter that conclusion. Patients presenting with EMD clearly appear to have higher risk disease with more proliferative cells and high-risk cytogenetic abnormalities. Disclosures:No relevant conflicts of interest to declare.

Indus river estuary: an assessment of potential risk of contaminants and ecosystem susceptibility

The Indus River is proclaimed as second most plastic-polluted rivers of the world. This river is the principal river of Pakistan and supplies freshwater for agriculture and human consumption. Its terminus into the northern Arabian Sea creates a unique ecosystem that supports a variety of aquatic organisms. In this study we have evaluate the heavy metal concentration in fishes sampled from the IRE. Muscle tissues from five fish species of ecologically and economically important were sampled and the concentrations of cadmium (0.125‒1.025 µg g−1, 0.93 ± 0.33), lead (0.250‒2.560 µg g−1, 0.92 ± 0.86), arsenic (4.178‒6.337 µg g−1, 4.24 ± 2.13) and mercury (BDL‒0.116 µg g−1, 0.05 ± 0.04) were found to be beyond optimum level. We determined the pollution load index which indicated that the IRE pollution exhibits significant seasonal oscillations. In addition to the heavy metal assay we note the frequent appearance of abnormal fishes caught in the IRE, which validates the pollution load. Multivariate approaches, canonical correspondence analysis and cluster analysis, were used to evaluate the relationships among environmental variables that influence metal concentration. This study is the first to document heavy metals detected from fishes inhabiting in IRE and highlights concerns regarding the need for management measures.HighlightsWe examined heavy metals in commercial and ecological valuable fishes in the Indus River Estuary (IRE) for the first time.Multivariate approaches were used to determine the efficacy of environmental parameters to predict heavy metal concentration.We described the presence of fish abnormalities as a result of contamination in the IRE.We observe that the mechanism of action of organism health and food contamination in the IRE is poorly understood.

Microdeletion in Chromosome 17 Cytoband q21.31 Identified As Located within the KANSL1 Gene Prevailed in Flt3-ITD Negative Normal Karyotype AML Patients As Opposed to Their AML Counterparts and Influenced the Phenotype of the Disease

Recent advances in genetic tools for leukemic diagnosis have facilitated studies on genetic traits which in concert with a driving mutation if present may influence the phenotype of the disease. Flt3-ITD is present in about 30% of AML patients and tends to be associated with a poor prognosis. Currently the use of inhibitors of mutated and in consequence activated kinases is undergoing clinical trials. The dim prognosis of Flt3-ITD positive AML and the hope associated with kinases inhibitors prompted us to evaluate in some depth the genetics of AML cases in relation to the presence of Flt3-ITD .Ninety-two AML patients (diagnosed according to the FAB/WHO criteria, F/M=49/43, age median: 57, range: 21-81 years) who were seen in our institution were recruited to the present study which was conducted as follows:The patients underwent GTG karyotyping and/or FISH for X or Y deletion, inv (3), -5/5q-, -7/7q-,+8, MLL, RUNX1, PML/RARA or RARA, inv(16). 52 lacking GTG and/or FISH abnormalities were called as normal karyotype (NK) patients, among those, 20 were Flt3-ITD positive. The other 40 patients had karyotype abnormalities or were FISH positive for AML-associated abnormalities among which 10 had Flt3-ITD .In all 92 patients microarray analysis was performed using Agilent - Catalog Agilent Cancer CGH+SNP 180K or Roche - WG Catalog NimbleGene 12x270K microarrays.We found (Partec Genomics Suite and Biodiscovery Nexus 8 software) that NK Flt3-ITD negative had, when the whole genome was analyzed, a higher number of total copy number variation (CNV) aberrations (amplifications and/or deletions, vs 32.21±8.70 vs 16.15±2.60, p=0.008) including one copy losses, (2.79±0.57 vs 1.20±0.35 p=0.019)as opposed to NK patients having Flt3-ITD mutation .During the study, an interim analysis was performed at the time we switched from Roche to Agilent microarrays which revealed the significant prevalence of CNVs in AML Flt3-ITD negative patients over Flt3-ITD positive ones within the region 17q21.31 specifically in the positions from 44281092 to 4476549, 44702953 to 44745272 and 44745272 to 44765496. The same region appeared again as significantly discriminating Flt3-ITD negative and positive patients at the final analysis. Therefore, this region was analyzed in some depth. Hg19 annotation was used and according to that the region encompasses the KANSL1 gene among two others (NSFP1 and NSF). The NSF gene encodes a protein involved in transport to the Golgi and NSFP1 is a pseudogene. KANSL1, a component of the MLL1 complex (thought to contribute to leukemogenesis), was the focus of our interest, and the gene was further investigated to validate the microarray-based platform results and to localize the CNV area more precisely. In the region 44281092 to 4476549 within the KANSL1 gene Flt3-ITD negative patients had more deletions and amplifications as compared to those Flt3-ITD positive (31/62 vs 7/30, p=0.012). The same was valid when only deletions were considered (19/62 vs 4/30, p=0.058).Then to validate microarray platform results the same 80 samples were examined using the TaqMan Copy Number Assay (ThermoFisher) for the KANSL1 gene (primers and probe designed for the region 44297111-44297201 lying in chr17), and as a reference the RNaseP gene was employed. The results obtained using the microarray platform correlated well (r=0.671, p<0.001) with those obtained using RT-PCR copy number assay for KANSL1 .61 DNA samples obtained from the patients' solid organ biopsies were examined using the Copy Number Assay for KANSL1 gene CNV. In all instances except one the same CNP was observed in the marrow as that in the solid organ biopsies. It lends credit to the technology employed in this study, but still we do not have enough data to exclude the somatic origin of the microdeletions.The microdeletions are of biologic significance, as suggested by the following findings: 1.All three patients with FISH-documented MLL amplification had deletions within the KANSL1 gene (chr:17 44281092 to 4476549).2.Microdeletions within the KANSL1 gene were more frequently observed in the group M0-M2 patients than in other subtypes of AML (18/51 vs 5/35, p=0.026).In conclusion, a lack of Flt3-ITD and normal karyotype was associated with the frequent presence of microdeletions within the chr17 cytoband q21.31. The latter was identified as (i) present within the KANSL1 gene and (ii) influencing the phenotype of AML.Supported by the INNOMED/I/1/NCBR/2014 DisclosuresNo relevant conflicts of interest to declare.

Standardized Targeted Sequencing of 29 Genes with Recurrent Mutations in Chronic Lymphocytic Leukemia (CLL) Helps to Better Define Clinical Correlates and Genomic Hotspots

Standardized Targeted Sequencing of 29 Genes with Recurrent Mutations in Chronic Lymphocytic Leukemia (CLL) Helps to Better Define Clinical Correlates and Genomic Hotspots

Gene Expression Profiling Is a Strong Predictor of Clinical Outcomes in Newly Diagnosed Multiple Myeloma

Introduction: Gene expression profiling (GEP) of purified CD138+ bone marrow plasma cells (PC) in patients with newly diagnosed multiple myeloma (MM) has proven to be a reliable prognostic tool. In this study, we aimed to describe the clinical outcomes of all consecutive NDMM patients with available GEP pre-treatment and compare GEP with standard FISH in determining relapse free survival (RFS) and overall survival (OS).Methods: From April 2014 until January 2017, we identified 104 patients with NDMM who had baseline GEP70 analysis at our institution. GEP70 was performed using CD138+ cells from bone marrow samples at diagnosis through MyPRS® (Signal Genetics, Little Rock, AR). Fisher's exact test was used to evaluate the associations between two categorical variables. The Wilcoxon rank sum test was used to evaluate the difference in continuous variables between patient groups. Kaplan-Meier method was used to estimate the time to event endpoints including RFS and overall survival (OS). The log-rank test was used to evaluate the differences in the time to event endpoints between different risk group categories. Univariate Cox proportional hazards model was used to evaluate the association between a continuous variable and RFS/OS. Statistical software SAS 9.3 (SAS, Cary, NC) and S-Plus 8.2 (TIBCO Software INc., Palo Alto, CA) were used for all analysis.Results: Median age was 63 (37-89) (Table 1a). Patients presented with lytic lesions (78%), anemia (78%), kidney dysfunction (13%) and hypercalcemia (31%). One patient did not meet CRAB criteria or biomarkers of malignancy, but had severe recurrent infections attributed to underlying myeloma requiring initiation of systemic therapy which prompted resolution of infections. One patient was initiated on therapy based on an i:u sFLC ratio of 165. All patients with available data for initial treatment (n=101), were treated either with bortezomib (63%), carfilzomib (37%) in combination with lenalidomide (86%) or cyclophosphamide (14%) and dexamethasone (100%). All patients received at least a three-drug combination as initial treatment, with 73% of patients receiving an upfront autologous transplant. Upfront autoSCT improved RFS (n=9/66; 14%) compared to non- or delayed autoSCT (n=8/25; 32%; p=0.03) but did not affect OS (p=0.86). Two patients (2 %) proceeded to an upfront allogeneic transplant as part of a clinical trial. With up to 36 months of follow up (range 0.9-35.9 months), 74% of patients are relapse free and 86% of patients are alive (Table 1b). GEP risk category predicted RFS (p=0.0005) in this series of patients (Figure 1). Table 1c shows GEP risk subtypes with clinical outcomes and association to FISH abnormalities. Uniformly, patients with high risk FISH abnormalities and low risk GEP had better clinical outcomes than patients with high risk GEP. High risk GEP patients represented 20% of all NDMM in this cohort. At one year, about 50% of these patients had relapsed or died compared to only 5% of patients in all of the other categories of GEP. By FISH, only t(14;16) predicted RFS (p=0.0001). Neither FISH nor GEP significantly predicted OS (P>0.05) although there were trends for worse survival for high risk GEP and FISH t(14;16).Conclusion: In this cohort of NDMM patients treated with modern therapies with most patients having received an autoSCT, GEP risk profiling was one of the best predictors of RFS. Longer follow up will be needed to determine prediction of OS in this cohort of patients. [Display omitted] DisclosuresLee:Eutropics Pharmaceuticals: Research Funding; Adaptive: Membership on an entity's Board of Directors or advisory committees; Pimera Inc: Consultancy; Celgene: Consultancy; Takeda: Consultancy; Daiichi Sankyo: Research Funding. Patel:Pfizer: Membership on an entity's Board of Directors or advisory committees, Research Funding; Juno: Consultancy; Celgene: Consultancy. Thomas:Celgene: Research Funding; Bristol Myers Squibb: Research Funding. Neelapu:Merck: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Bristol-Myers Squibb: Research Funding; Celgene: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Karus: Research Funding; Cellectis Inc.: Research Funding; Kite Pharma: Consultancy, Membership on an entity's Board of Directors or advisory committees, Research Funding; Novartis: Consultancy, Honoraria, Membership on an entity's Board of Directors or advisory committees; Poseida Therapeutics, Inc: Research Funding. Orlowski:BioTheryX: Consultancy, Membership on an entity's Board of Directors or advisory committees. Manasanch:adaptive biotechnologies: Consultancy; quest diagnostics: Research Funding; sanofi: Research Funding; merck: Research Funding; celgene: Consultancy; takeda: Consultancy.

Fluorescence in Situ Hybridization (FISH) Frequently Upstages Risk Estimates for MDS Patients with Normal Metaphase Karyotype

IntroductionThe international prognostic scoring system (IPSS) and its revised version (R-IPSS) are the most widely used clinical models for MDS risk stratification. Both models account for cytogenetic abnormalities captured by G-banding metaphase karyotyping. Among MDS patients, 50-60% will have a normal karyotype. MDS FISH panels are commonly applied to supplement cytogenetics. In small subsets of patients with normal karyotype, MDS FISH may detect cytogenetic abnormalities not resolved by metaphase karyotype. The utility of MDS FISH panel in refining IPSS or R-IPSS cytogenetic risk score among those patients is not known. We explored the impact of FISH in upstaging MDS patients with a normal karyotype and its prognostic value if incorporated into IPSS or R-IPSS cytogenetic risk score.MethodsWe identified patients with normal karyotype by G-banding who had cytogenetic abnormalities detected by an MDS FISH panel. The MDS FISH panel included probes for deletion (del(5)), del(7), del (20), trisomy 8 and del 17p. IPSS and R-IPSS was calculated based on karyotype as the standard recommendation and then recalculated according to cytogenetic abnormalities detected by the MDS FISH panel (FISH_IPSS and FISH_R-IPSS). Median overall survival (OS) was calculated from time of diagnosis using Kaplan-Meier estimates.ResultsAmong 1600 patients in our MDS database with normal karyotype by G-banding, 53 patients (3%) had a cytogenetic abnormality detected by FISH. The cytogenetic abnormalities detected included del(7)/monosomy 7 in 25/53 patients (47%), del(5q)/monosomy 5 in 22/53 patients (42%), trisomy 8 in 14/53 (26%), del(20q) in 10/52 (19%), and 17p deletion in 1/10 (10%).According to IPSS, 15 patients (33%) were classified as low risk, 18 patients (40%) as intermediate-1 (int-1), 9 patients (20%) int-2, and 3 patients (7%) as high risk. Table-1 summarizes restaging using FISH_IPSS where 53% of low risk IPSS were upstaged to int-1, 56% of int-1 upstaged to int-2, and 78% of int-2 upstaged to high risk.By conventional R-IPSS, 18 patients (40%) were very low risk, 11 patients (24%) low risk, 12 patients (26%) intermediate risk, and 4 patients (9%) high risk. Upon restaging using FISH_R-IPSS (Table 2), 61% of very low risk patients, all low risk patients, 92% of intermediate risk patients, and 50% of high risk patients with FISH abnormalities were upstaged.Among patients with low risk IPSS, median OS was 64 months (mo) for those low risk by FISH_IPSS and 61 mo for those upstaged to int-1 by FISH_IPSS. For patients with int-1 risk IPSS, median OS was 26 month for those int-1 by FISH_IPSS and 24.8 mo for those upstaged to int-2 risk by FISH_IPSS. Finally, for patients with int-2 risk IPSS, median OS was 30 mo for those classified as int-2 and 22 mo for those upstaged by FISH_IPSS.Based on R-IPSS the median OS was 64, 24, 25 and 19 mo for very low, low, int, and high risk groups. Using FISH_RIPSS the median OS was 64, 61,30,21,22 mo respectively for very low, low, int, high and very high risk groups (p.003). Based on R-IPSS cytogenetic risk category all those patient were classified as good risk given normal karyotype with median OS of 30 mo. Based on FISH data if cytogenetic score was re-calculated accordingly, 16 patients remained good risk, 22 patients intermediate risk, and 14 patients high risk. The median OS was 30, 30 and 22 mo respectively.ConclusionsFISH detected cytogenetic abnormalities in a small number of patients with normal karyotype by G-banding, which altered risk score assignment by IPSS and R-IPSS in a significant subset of positive patients. [Display omitted] DisclosuresKomrokji:Celgene: Honoraria; Novartis: Honoraria, Speakers Bureau. Sallman:Celgene: Research Funding. Padron:Incyte: Honoraria, Research Funding. Sweet:Ariad: Consultancy, Speakers Bureau; Karyopharm: Consultancy, Research Funding; Otsuka: Consultancy; Incyte: Research Funding; Pfizer: Consultancy; Novartis Pharmaceuticals: Consultancy, Speakers Bureau. Lancet:Pfizer: Other: Institutional research funding, Research Funding; Boehringer Ingelheim: Consultancy; Novartis: Consultancy; Celgene: Consultancy; Erytech: Consultancy; BioSight: Consultancy; Bio-Path Holdings: Consultancy; Janssen: Consultancy; Jazz Pharmaceuticals: Consultancy.

Clonal Evolution in CLL

Clonal Evolution in CLL