Cytogenetic Karyotype Research Articles

Molecular cytogenetic analysis of genome-specific repetitive elements in Citrus clementina Hort. Ex Tan. and its taxonomic implications

BackgroundClementine mandarin (Citrus clementina Hort. ex Tan.) is one of the most famous and widely grown citrus cultivars worldwide. Variations in relation to the composition and distribution of repetitive DNA sequences that dominate greatly in eukaryote genomes are considered to be species-, genome-, or even chromosome-specific. Repetitive DNA-based fluorescence in situ hybridization (FISH) is a powerful tool for molecular cytogenetic study. However, to date few studies have involved in the repetitive elements and cytogenetic karyotype of Clementine.ResultsA graph-based similarity sequence read clustering methodology was performed to analyze the repetitive DNA families in the Clementine genome. The bioinformatics analysis showed that repetitive DNAs constitute 41.95% of the Clementine genome, and the majority of repetitive elements are retrotransposons and satellite DNAs. Sequential multicolor FISH using a probe mix that contained CL17, four satellite DNAs, two rDNAs and an oligonucleotide of (TTTAGGG)3 was performed with Clementine somatic metaphase chromosomes. An integrated karyotype of Clementine was established based on unequivocal and reproducible chromosome discriminations. The distribution patterns of these probes in several Citrus, Poncirus and Fortunella species were summarized through extensive FISH analyses. Polymorphism and heterozygosity were commonly observed in the three genera. Some asymmetrical FISH loci in Clementine were in agreement with its hybrid origin.ConclusionsThe composition and abundance of repetitive elements in the Clementine genome were reanalyzed. Multicolor FISH-based karyotyping provided direct visual proof of the heterozygous nature of Clementine chromosomes with conspicuous asymmetrical FISH hybridization signals. We detected some similar and variable distribution patterns of repetitive DNAs in Citrus, Poncirus, and Fortunella, which revealed notable conservation among these genera, as well as obvious polymorphism and heterozygosity, indicating the potential utility of these repetitive element markers for the study of taxonomic, phylogenetic and evolutionary relationships in the future.

Cytogenetic investigation of couples with recurrent spontaneous miscarriages.

Background & Objective:Spontaneous pregnancy loss has always been the frustrating experience for the couples and concern clinician. Chromosomal abnormality in either of the parent is considered to be the one of the leading cause of recurrent spontaneous miscarriages. This study was designed to evaluate the possible chromosomal etiology of miscarriage and the subsequent intimacy of maternal or paternal genetic abnormality.Methods:This case-control study was conducted between January 2016 and October 2016 at a tertiary care hospital in Karachi. A total of thirty-two couples were selected who had suffered with recurrent spontaneous miscarriages (RSM). Using conventional cytogenetic technique karyotyping was performed on all of the subjects. For the control twenty couples were also selected with no history of pregnancy loss. All the karyotypes were recorded on the standard method. Data was analyzed through SPSS version 22.Results:Among thirty-two cases nine cases were found to have abnormal karyotype. In which sex chromosomal trisomy=02 (46,XY/47,XXY), marker chromosome=01 (47,XX,+mar), Robertsonian translocation=01 (45,XY,der,(14:21),(q10;q10)), reciprocal translocation=01 (46,XX,t(11;22)(q23;q11.2)), inversion=02 (46,XX,inv(9)(p11q13)) and minor structural abnormalities=02 (46,XX,15PS+) were found. Approximately equal ratio with 1:1.25 was observed among male and female carrier respectively. Non-significant difference was found between the ages of both carriers (p=0.34). Though a significant different value was calculated in the case of number of miscarriage (p=0.004*). Moreover, no significant association was found among spontaneous miscarriage (SM) and recurrent spontaneous miscarriage (RSM) with respect to maternal age (p= 0.157).Conclusion:In the recent study possible chromosomal abnormalities suggested the evaluation of the patient with the history of recurrent spontaneous miscarriage must include conventional cytogenetic. Moreover, probe development and extended investigation can ease the prognosis among pregnancy related complication.

Cytogenetic effects of gestogens on women with threatened miscarriage in Duhok Province, Kurdistan Region, Iraq

Background: Gestogens have been recommended to have a place in the prevention of threatened miscarriage and spontaneous pregnancy loss during the first 3 months of pregnancy. Certain studies consider that this steroid hormone has genotoxic effect because they directly involved in the change of DNA structure. Objectives: This study aims to investigate the cytogenetic effects of gestogens on peripheral blood lymphocytes of pregnant women, by damaged cells and chromosomal aberrations, as well as evaluate the models for hormonal therapy in vivo. Materials and Methods: Blood sample from 30 women with threatened miscarriage, who received gestogen therapy in the first 3 months, Were taken. Cytogenetic analyses and karyotyping were performed for Each patient Before and after therapy. Results: The numerical chromosomal aberrations which found in blood lymphocytes of women before taking gestogens were 44,XX and 48,XX. The same result was observed after taking gestogens. The percentage levels of structural chromosomal aberrations were ring chromosome (2.06%), chromatid breaks (12.47%), chromatid gaps (1.65%), dicentric (0.55%), acentric chromosome (4.02%), and interchange chromosome (12.5%). Whereas, the abnormalities after treatment with gestogens were ring chromosome (5.31%), chromatid breaks (42.19%), chromatid gaps (20.86%), dicentric (7.50%), acentric chromosome (17.75%), and interchange chromosome (43.97%). Conclusion: Gestogens as hormonal substitute therapy do not induce numerical chromosomal aberrations but have a significant increase of structural chromosomal aberrations Gestogens have genotoxic effects on human lymphocyte chromosomes at wide range of concentrations. So the potential benefits and side effects of it must be weighed up against the deleterious effects.

Discrepant Mutational Composition between Myeloid Sarcoma and Bone Marrow Leukemia Revealed through Targeted Next Generation Sequencing

Introduction:Myeloid sarcoma (MS) is the involvement of acute myeloid leukemia (AML) cells in extramedullary tissues, whose mechanism remains to be further elucidated. How MS affects AML prognosis at a molecular level is also an open question. This unexplained tropism of leukemic blasts to extramedullary tissues is likely caused by the combination of genetic and epigenetic aberrations. Our study investigated the paired samples of myeloid sarcoma tissue and bone marrow (BM) at diagnosis in perspective of elucidating specific genetic abnormalities that underpin the ectopic homing of MS cells.Methods:A 62-patient cohort of pathologically-proven MS diagnosed between 2005-2018 was systemically reviewed. Histomorphology and immunophenotypes of all cases were reviewed by the hematopathologist. Cytogenetic karyotyping in BM cells was carried out with conventional G-banding method. Twenty-five cases of extramedullary presentation at initial diagnosis were further interrogated with next generation sequencing. DNA was extracted from formalin-fix-paraffin-embedded (FFPE) tissue using an error-avoiding reagent against formaldehyde-caused artificial single nucleotide changes (QIAGEN GeneRead). Mutations in 54 genes associated with myeloid disorders were analyzed in Illumina MiSeq sequencer along with the TruSight Myeloid Sequencing Panel. FLT3-ITD in bone marrow was detected by Sanger sequencing. Of these, 16 patients' paired samples (16 FFPE tissue and 16 BM tissue) were analyzed in comparison.Results:MS occurred concomitantly with leukemic marrow in 21 patients, without bone marrow disease in 2 patients, and resulted from transformation of MPN or MDS/MPN in 2 patients. The median age of diagnosis is 45 years (range 17-72 years), and the male to female ratio is 1.28. Twenty patients received standard treatment with systemic chemotherapy, and nine patients underwent subsequent allogeneic transplantation. The median overall survival was 31.6months ± 20.83 months with a median follow up of 28 months. Cytogenetic patterns included 9 patients with normal karyotypes, 5 with complex changes and 4 with a core-binding factor aberration. The median of mutation numbers in MS was three (range 0- 5) per case. In agreement with prior reports, the most common molecular abnormality in MS was NPM1 (11/25, 44%), followed by FLT3-ITD (7/25, 28%) and DNMT3A (6/25, 24%). The coexisting genetic alterations in the BM from patients without evident AML in morphology or immunophenotypic analyses, suggesting a role for clonal hematopoiesis in the BM. All of the cases with RUNX1-RUNX1T1 translocation harbored KIT mutation. Nineteen out of 25 cases (76%) had a mutation in the genes of receptor tyrosine kinase (RTK)- RAS pathway. In MS-BM paired samples, additional driver mutations in the MS samples included DNMT3A, IDH1, ASXL1, KRAS, CBL, NPM1, KIT and U2AF1. Conversely, one patient's BM PTPN11 mutation was lost in his MS lesion. For more details, the cytogenetic changes, mutational profile and the variant allele frequency were summarized in Figure 1.Conclusion:Interestingly, additional driver mutations were identified in the MS samples compared to the BM counterparts, suggesting that a comprehensive characterization of AML heterogeneity might need the mutational profiling of the coexisting MS. AML patients with MS infers a negative prognosis and were treated with frontline allogeneic stem cell transplantation whenever possible in this study. Consistent with prior report, this study showed a higher incidence of genetic alteration of NPM1, FLT3-ITD and DNMT3A in MS along with an enrichment of RTK-RAS signaling pathway mutations. Functional study is needed to delineate the biology underlying the ectopic homing of these commonly mutated genes in AML.(Figure legend)Molecular signature of 25 patients with myeloid sarcoma. Highlight in red represents pathologic mutation, highlight in blue represents mutation of unknown significance. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Mubritinib Targets the Electron Transport Chain Complex I and Reveals the Landscape of Mitochondrial Vulnerability in Acute Myeloid Leukemia

BACKGROUND60% to 70% of Acute Myeloid Leukemia (AML) patients enter complete remission after induction regimen, but the majority relapse within 3 years due to the outgrowth of therapy resistant Leukemia Stem Cells (LSCs). Identification of novel treatment strategies effective against these cells thus represents an outstanding medical need. We developed a cell culture method, which transiently maintains LSC activity ex vivo (Pabst et al., Nature Methods, 2014) and enables chemical interrogation of cell types relevant for the progression of the disease. Overall, HSCs and LSCs share numerous biological traits, making specific LSC eradication challenging. However, striking differences in energy metabolism between normal and leukemic stem cells have recently been suggested. While HSCs appear to rely primarily on anaerobic glycolysis for energy production, LSCs seem to depend on mitochondrial oxidative phosphorylation for their survival. Targeting mitochondrial respiration could therefore represent an effective approach for the specific eradication of LSCs. AIMWe aimed to identify novel therapeutic targets for AMLs with poor treatment outcome. The study relied on the Leucegene approach that integrates results generated by RNA sequencing analysis of primary human AML specimens, detailed clinical and cytogenetic annotations provided by the Quebec leukemia cell bank and ex vivo responses of primary AML samples to various chemical compounds. Our study specifically focused on specimens originating from patients with poor (overall survival < 3 years) and good (overall survival ≥ 3 years) response to standard chemotherapy, and did not include cases of Acute Promyelocytic Leukemia (APL). RESULTSWe identified Mubritinib, previously described as an ERBB2 inhibitor, as a novel anti-leukemic agent, which selectively inhibits the viability of leukemic cells from therapy-resistant AML patients, but does not affect normal CD34+ cord blood cells. Exposure to Mubritinib triggered apoptotic cell death in a subset of AML samples with high mitochondrial function-related gene expression, high relapse rates, and short overall survival. Sensitivity to Mubritinib also strongly associated with the intermediate cytogenetic risk category, normal karyotype (NK), and NPM1, FLT3 (ITD) and DNMT3A mutations. Conversely, resistance to Mubritinib associated with favorable cytogenetic risk AMLs, Core Binging Factor (CBF) leukemias and KIT mutations. Mubritinib has been developed as an ERBB2 kinase inhibitor. Intriguingly, we found that ERBB2 is not expressed in Mubritinib-sensitive AML specimens, suggesting that the anti-leukemic activity of this compound is likely not mediated by ERBB2 inhibition. Using a combination of functional genomics and biochemical analyses, we demonstrated that Mubritinib directly inhibits the mitochondrial Electron Transport Chain (ETC) complex I, which leads to a decrease in oxidative phosphorylation activity and to induction of oxidative stress. The impact of Mubritinib on AML progression was explored using a syngeneic mouse model (MLL-AF9 tdTomato-positive leukemia). Recipients of MLL-AF9 cells treated with Mubritinib exhibited a 19-fold decrease in the number of tdTomato-positive cells in the bone marrow and a 42-fold decrease in the spleens compared to control mice. Short-term treatment also led to a 37% increase in the median overall survival of Mubritinib exposed recipients compared to vehicle treated mice. Importantly, and in agreement with our observation that Mubritinib treatment does not impede proliferation of normal hematopoietic CD34+ cells in vitro, Mubritinib treatment had no impact on the number of non-transduced (tdTomato negative) nucleated bone marrow cells of recipients. CONCLUSIONSWe uncovered the clinical, mutational, and transcriptional landscape of mitochondrial vulnerability in AML and identified Mubritinib as a novel ETC complex I inhibitor with therapeutic potential for approximately 30% of AML cases currently lacking effective treatment options. As Mubritinib completed a phase I clinical trial in the context of ERBB2-positive solid tumors, our work suggests an opportunity to re-purpose Mubritinib's usage for this genetically distinct subgroup of poor outcome AML patients. DisclosuresNo relevant conflicts of interest to declare.

Marker Chromosomes Are a New Cytogenetic Adverse Risk Factor in AML after Allo-HCT

[Background]Chromosome analysis is indispensable for the diagnosis and risk classification of acute myeloid leukemia (AML). A marker chromosome (MC) is a fragmented chromosome that cannot be identified as originating from an existing autosomal or sex chromosome. Although often observed, the significance of MC in hematological malignancies is unknown. Recently, MC was found to be the result of chromothripsis (CT). CT is a catastrophic phenomenon by which chromosomes are shattered into tens to hundreds of fragments. Half of all CT cases are related to TP53 mutation. In addition to MC, complex karyotype (CK), monosomal karyotype (MK) and existing sub-clone (SC) have also been noted as the result of CT. Previous studies reported that MC was a poor prognostic factor in AML. These studies included AML patients who underwent allogeneic hematopoietic cell transplantation (allo-HCT). However, the influence of MC on AML after allo-HCT was not clarified.[Purpose]In this study, we evaluated the impact of MC on the outcome of AML patients after allo-HCT.[Method]This retrospective analysis included 166 AML patients who received allo-HCT at Niigata University Hospital (n=129) or Nagaoka Red Cross Hospital (n=37) between 1990 and 2017. The median age of patients at allo-HCT was 38 years old (range 14-67 y). The median follow-up period was 2.0 y (range 0.0-22.2 y). According to the revised Medical Research Council (rMRC) criteria for cytogenetic risk categories, 26 (15.7%), 104 (62.7%) and 36 (21.7%) patients were categorized as favorable, intermediate and adverse-risk, respectively. Myeloablative conditioning was used for 128 (77.1%) and reduced-intensity was used for 38 (22.9%) patients. Donors were related for 83 (59.3%) and unrelated for 57 (40.7%) patients. The Kaplan-Meier method (log-rank test) and Gray's test were used to estimate the probabilities of overall survival (OS) and cumulated incidence of relapse (CIR). The impact of several variables was assessed by multivariate analysis using a Cox regression model and Fine-Gray test with backward stepwise selection based on the p-value. For the comparison of clinical phenotypes, category variables were evaluated by Fisher's exact test.[Results]MC was detected in 14 (8.4%) of 166 patients. Eleven (78.6%) were grouped as adverse-risk by rMRC criteria. CK, MK and SC were observed in 26 (16.3%), 20 (12.0%) and 23 (13.9%) patients, respectively. The median age of AML/MC+ (n=14) and AML/MC-(n=152) patients were similar (38.5 y, range 19-64 y vs 38 y, range 14-64 y, P=0.812). Twelve AML/MC+ patients (85.7%) received allo-HCT at the advanced stage (³3 CR or non-remission) and 10 (71.4%) had primary induction failure (PIF). Compared with AML/MC- patients, those with AML/MC+ had a higher incidence of MK (78.6% vs. 5.9%, p<0.001), sub-clone (64.3% vs. 9.2%, p<0.001) and CK (85.7% vs. 9.9%, p<0.001). On univariate analysis, the 2-y OS and median survival period of AML/MC+ patients were shorter than those of AML/MC- patients (26.8% vs. 61.3% and 0.78 y vs. 4.9 y, p=0.0126). The 2-y CIR of AML/MC+ patients was higher than that of AML/MC- patients (80.4% vs. 37.2%, p<0.01). To further investigate the impact of MC on the outcome, we compared AML patients with/without MC who had the cytogenetic adverse-risk karyotype. The 2-y OS of adverse-risk AML/MC+ patients (n=11) was shorter than that of adverse-risk AML/MC- patients (n=25) (9.1% vs 58.3%, p=0.003). The median survival periods were 0.58 y and 4.0 y, and the 2-y CIR was 89.6% and 44.7% (p=0.002), respectively. The 2-y OS based on the other risk factors in adverse-risk AML patients were as follows: CK+ 33.2%, MK+ 26.9% and SC+ 34.7%. As these results suggested MC to be a poorer risk factor, we performed multivariate analysis for confirmation. In the multivariate analysis adjusted for CK, MK, SC, donor, conditioning and disease stage before allo-HCT, only MC was an independent poor risk factor for OS (HR 3.547, 95%CI: 1.46-8.63, p=0.005) and CIR (HR 3.90, 95%CI: 1.47-10.33, p=0.006) among adverse-risk AML patients using the rMRC criteria.[Conclusion]It is very difficult for AML/MC+ patients to achieve complete remission, leading to a markedly poor prognosis after allo-HCT. The outcome for AML/MC+ was inferior to that for AML with CK, MK, SC or the current adverse-risk karyotype by rMRC. This analysis revealed MC as a new independent factor that further indicates a poor prognosis after allo-HCT, especially in high-risk AML patients. DisclosuresNo relevant conflicts of interest to declare.

A Next-Generation Sequencing-Based Karyotyping Algorithm Reveals the Genomic Structure of Acute Myeloid Leukemia

Introduction: The BeatAML master trial is evaluating the use of precision medicine via genomically guided therapy in acute myeloid leukemia (AML). A variety of assays are used to test patient samples including cytogenetic karyotyping, fluorescence in situ hybridization (FISH), and next-generation sequencing (NGS)-based approaches to analyze genomic alterations. We developed an algorithm to extract karyotype information from NGS-based genomic sequencing data in the BeatAML cohort. We also utilized this algorithm to retrospectively study 1603 unique AML samples collected during routine clinical care.Methods: Comprehensive genomic profiling (CGP) was performed using a CLIA/CAP/NYS approved DNA and RNA hybrid capture-based next-generation sequencing assay that sequences 426 genes in DNA and 265 genes to capture fusions by RNA. We analyzed aneuploidy by comparing the log ratio of read counts in tumor DNA to a process matched normal control and developed signal to noise metrics to measure chromosome arm copy number. A chromosome arm was considered altered if >50% of the arm was altered. Chromosome (chr) 5q was also considered lost if only chr5q31 (containing EGR1) was lost. Based on the noise metrics of each sample, we also calculated a per sample limit of detection, which was defined as the lowest percentage of cells that could have a single copy gain or loss and be detected. We validated chr5q loss, chr7q loss, and chr8 gain using FISH results obtained from available BeatAML samples as the gold standard. To better understand the genomic landscape of AML, we also included 1603 samples run in standard clinical practice. False discovery rate (FDR) was assessed using the Bonferroni method.Results: We developed a method to analyze chr5q loss, chr7q loss and chr8 gain that had 95% concordance across all chromosomes compared to the FISH gold standard. The median limit of detection was 10.1% (percentage of cells required to have a single copy gain/loss to be detected). Overall concordance adjusted for limit of detection was 97% with 92% sensitivity (55/60 chromosomes) and 99% specificity (162/164 chromosomes) with similar performance across chromosomes. For chr7q loss and chr8 gain, over 50% of the chromosomal region was altered. However, the size of the loss on chr5q varied widely from the entire arm to a single gene (EGR1). We next applied the algorithm to 1603 unique patient samples analyzed by CGP during routine clinical care. We detected loss of chr5q in 10.9% (174), loss of chr7q in 10.4% (166), and gain of chr8 in 9.2% (147) of AML samples. Frequently altered genes in this cohort included FLT3 (21%, 337), RUNX1 (20.5%, 328), DNMT3A (17.5% 280), and TP53 (17.5%, 280). To further explore the genomic landscape, we analyzed the cooccurrence of each chromosomal alteration with all genes assayed. All three chromosomal aberrations exhibited significant cooccurrence with each other and TP53 (FDR P < 0.05). TP53 was altered in 75% (130/174) of chr5q loss samples 49% (82/166) of chr7q loss samples and 37% (55/147) of chr8 gain samples. Loss of chr5q also exhibited significant mutual exclusivities with FLT3, DNMT3A, NPM1, SRSF2, ASXL1, CEBPA, and WT1 (FDR P < 0.05). Loss of chr7q exhibited a slightly different mutual exclusivity profile with FLT3, NPM1, and PTPN11 (FDR P < 0.05). The difference in the cooccurrence profile between chr5q and chr7q was not a matter of p-value cutoffs since the magnitude of effect varied widely between groups.Conclusions: We demonstrated an NGS-based algorithm to determine karyotype that has a high concordance with FISH results. Limit of detection was equivalent to karyotyping by metaphase analysis but slightly worse than FISH assays on freshly prepared samples. We identified a previously described genomic stratification of TP53-aneuploidy since chromosomal aberrations and TP53 exhibited significant cooccurrence. However, only 50% of samples with loss of chr5q exhibited loss of chr7q and vice versa. While both were mutually exclusive with FLT3 and NPM1, loss of chr5q was mutually exclusive with several genes of prognostic significance including chromatin factors (DNMT3A) and splice factors (SRSF2). This study shows how NGS-based approaches can capture both chromosome arm level copy number alterations as well as all four categories of genomic alterations, thereby enabling risk stratification and identification of potential treatment options as defined in current NCCN guidelines. DisclosuresPavlick:Foundation Medicine Inc: Employment. Montesion:Foundation Medicine Inc: Employment. Severson:Foundation Medicine Inc: Employment. Brennan:Foundation Medicine Inc: Employment. Frampton:Foundation Medicine Inc: Employment, Other: Employee and stockholder. He:Foundation Medicine Inc: Employment. Levine:Roche: Consultancy, Research Funding; Epizyme: Patents & Royalties; Celgene: Consultancy, Research Funding; Isoplexis: Equity Ownership; Prelude: Research Funding; Qiagen: Equity Ownership, Membership on an entity's Board of Directors or advisory committees; Gilead: Honoraria; Janssen: Consultancy, Honoraria; Novartis: Consultancy; C4 Therapeutics: Equity Ownership; Imago: Equity Ownership; Loxo: Consultancy, Equity Ownership. Vergilio:Foundation Medicine Inc: Employment. Albacker:Foundation Medicine Inc: Employment; Foundation Medicine Inc: Employment.

Matrix Metalloproteinase and Tissue Inhibitor of Metalloproteinases Is Associated with Multiple Myeloma Progression, Prognosis and Extramedullary Plasmacytoma

Backgrounds and Aims: Matrix metalloproteinase (MMP) is endopeptidase enzyme degrading extracellular matrix, and tissue inhibitor of metalloproteinases (TIMP) is negative regulator of MMP. MMP is well known to be involved in metastatic mechanism of cancer cell and oncogenesis. However expression and role of MMP and TIMP has not been well established in multiple myeloma (MM). Therefore we examined whether expression of MMP and TIMP was involved in progression and prognosis of MM and extramedullary plasmacytoma (EMP) formation.Materials and Methods: Purified bone marrow plasma cells by using anti-CD138 antibody and magnetic beads obtained from 151 MM, 64 MGUS, 18 control and 5 EMP were subjected to the study after informed consent. The study was approved by IRB following Declaration of Helsinki. Whole transcriptome by next generation sequencer (NGS) using Illumina Next Seq 500 was performed in part of the samples to select genes to be studied, then expression level of MMP and TIMP determined by RQ-PCR Delta Ct value normalized with ACTB was used for analysis. MM cell lines KMS11, KMS12PE, KMS12BM, KMM1, RPMI8226, MM1S were used for the in vitro study.Results: We selected TIMP-1, 2and MMP14, 24 based on transcriptome analysis data comparing MM and EMP. The expression level of TIMP1 and MMP24 was significantly higher in MM (median delta Ct: 0.033 for TIMP1, 0.00025 for MMP24) than in MGUS (median delta Ct: 0.013 for TIMP1, 0.00006 for MMP24) (p=0.005, p=0.001), however TIMP2, MMP14 level did not differ in between MM and MGUS. Interestingly, TIMP1, 2 and MMP14, MMP24 were expressed with strikingly higher levels in EMP than in MM with 20 times, 60 times, 300 times, 500 times respectively (p=0.01, p=0.02, p=0.004, p=0.004). Both TIMP1 and 2 expression were higher in the MM patients with high risk cytogenetic karyotype t(4;14), t(14;16), del 17p than in the patients without such karyotype (p=0.006, p=0.008), however the levels of MMP14 and 24 did not differ in between cytogenetic risk groups. Positive correlations were found in between TIMP1 and 2, MMP14 and 24 in both MM and MGUS group respectively (r=0.34, p<0.001, r=0.49, p<0.001 in MM, r=0.32, p=0.008, r=0.63, p<0.001 in MGUS). Since TIMP2 and MMP14 were higher in EMP, the effect of recombinant TIMP2, siRNA-TIMP2 and MMP14 inhibitors marimastat/ilomastat were tested in MM cell lines KMM1, KMS11, KMS12PE and RPMI8226. However, these interventions did not change proliferation rates of these cell lines. The cell lines were treated with doxorubicin or bortezomib to study if these agent can change TIMP and MMP expression. Doxorubicin significantly increased expression level of MMP14, 24 and TIMP1, 2, but bortezomib increased only MMP14, 24. In the newly diagnosed MM (NDMM) patients (n=77), median time of overall survival (OS) of the patients with high TIMP1 (more than median value) was significantly shorter (2.7 years vs not reached) and 3 year OS rate was inferior (40% vs 65%) (p=0.0095). Progression free survival (PFS) tended to be inferior for high TIMP1 group, but the difference did not reach statistical significance (p=0.221). OS and PFS were not different according to either TIMP2, MMP14 or MMP24.Conclusions: Our results suggest that TIMP1, 2 and MMP14, 24 were associated with EMP formation. Among those factors, TIMP1 is the one which may play a key role for MM progression and chemo-resistance based on the results revealing its upregulation by antineoplastic agents and association with poor prognosis of MM patients. Our results is consistent with a previous report describing that high serum TIMP1 concentration was associated with poor prognosis of MM. TIMP is recently shown to play another role besides negative regulator for MMP, so further study to elucidate its specific role for chemo-resistance contributes to develop novel therapy targeting TIMP and MMP pathway. DisclosuresTsukamoto:Kyowa-Kirin: Research Funding; Chugai: Research Funding; Eisai: Research Funding; Pfizer: Research Funding. Handa:Celgene: Honoraria, Research Funding, Speakers Bureau; Takeda: Consultancy, Honoraria, Research Funding, Speakers Bureau.

Presentation Pattern, Diagnostic Markers, Management and Outcome of IgD Multiple Myeloma: A Systematic Review

Background:IgD multiple myeloma (MM) is a rare subtype contributing 2% of all multiple myeloma cases. Despite difficulty and delay in diagnosis, recent advances in the treatment of multiple myeloma in general also improved the outcomes for IgD myeloma subtype. The aim of this study is to summarize the data on presentation patterns, diagnosis, management approaches and outcomes for patients (pts) with IgD myeloma.Methods:A comprehensive literature search for articles published after December 2013 was performed using four databases: PubMed, Embase, Cochrane, and Clinicaltrials.gov. With initial search we identified 209 articles and after screening by two independent reviewers we included only 8 studies in the final analysis.Results:A total of 166 pts with IgD MM were included from 8 selected retrospective case series. Lambda (λ) was the predominant light chain sub-type in 136 patients (81.9%). Initial manifestations of IgD MM were Bence jones proteinuria (BJP>2 g/day-64.5%), renal dysfunction (63%), bone pain (55.9%), weakness, fatigue (34.2%) and extra medullary involvement (28.3%). Renal function was assessed by estimated glomerular filtration rate (eGFR<60 ml/min/1.73m2) in 54.3 % of patients and by serum creatinine level (Cr >2mg/dl) in 46.1% patients. Cytogenetic karyotype analysis in 56 pts using fluorescence in-situ hybridization (FISH) identified abnormal cytogenetics in 41 (73.2%) patients, cases were further classified as high risk (85.7%) and standard risk (14.3%). The patient characteristics and disease manifestations are mentioned in Table 1.Serum protein electrophoresis (SPEP) showed positive monoclonal spike (M) spike in 84% of the patients. The median M spike value for IgD MM was 9.42 g/l in a cohort of 17pts, (Djidjik et al.) which was lower as compared to IgG (median: 35 g/L) and IgA (median: 32 g/L) in this study. Abnormal serum free light chain ratio (sFLCR) was observed in 83% pts. Quantitative serum IgD levels were elevated only in 28% of the cases. Bone marrow (BM) plasmacytosis showing > 40% abnormal plasma cells was detected in 95.6% of the patients. N glycans are newly discovered biomarkers used for detecting abnormal protein glycosylation in MM patients. NG1(6)A2F and NG1(3)A2F were the two most significant N glycan markers for IgD MM patients with sensitivity of 95% & 95.2%, respectively and specificity of 95% & 78.6%, respectively. The prognostic significance of several other biomarkers studied in IgD MM is mentioned in Table 2.Treatment details were available for 149 patients and overall response rate (ORR) was seen in 125 (83.9%) patients while 24 (15.4%) patients had progressive or stable disease. The median overall survival (mOS) was between 9 - 62 months. Novel agents (NA) such as bortezomib, thalidomide and lenalidomide were given to 111 (74.4%) pts with the mOS between 15 - 38.6 months. Conventional agents (CA) such as melphalan, vinblastine, vincristine, epirubicin, and ifosfamide were given to 38 (25.5%) patients with mOS between 12.5 - 17 months. Data showed bortezomib based regimens resulted in a higher ORR (ORR=94%, CR=52%) compared to non bortezomib based regimens (ORR=77.8%, CR=27.7%) respectively. Stem cell transplant (SCT) was used in 37 (24.8%) patients with chemotherapy (NA=26, CA=11) which showed good response rate (ORR>90%,CR >60%). The comparison of response rate with different drugs is mentioned in the Table 3.Conclusion:N-glycan assay can overcome the limitations of SPEP and quantitative serum IgD assay in the diagnosis of IgD MM. This difficulty is posed by the lower concentrations of M spike ( SPEP) or lower-normal serum IgD value on quantitative serum assays, which can delay its diagnosis. Novel and conventional agents have good responses with ORR>90%, but mOS of conventional agents was lower when compared to novel agents. Bortezomib based regimens showed better responses when compared to non-bortezomib regimens. The best response (ORR=95%) was obtained with use of SCT in combination with novel agents. Serum free light chain levels, N glycan assay and serum IgD quantification were found to be the best prognostic markers for IgD MM. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Cytogenetics and gene mutations influence survival in older patients with acute myeloid leukemia treated with azacitidine or conventional care

Older patients with newly diagnosed acute myeloid leukemia (AML) in the phase 3 AZA-AML-001 study were evaluated at entry for cytogenetic abnormalities, and a subgroup of patients was assessed for gene mutations. Patients received azacitidine 75 mg/m2/day x7 days (n = 240) or conventional care regimens (CCR; n = 245): intensive chemotherapy, low-dose cytarabine, or best supportive care only. Overall survival (OS) was assessed for patients with common (occurring in ≥10% of patients) cytogenetic abnormalities and karyotypes, and for patients with recurring gene mutations. There was a significant OS improvement with azacitidine vs CCR for patients with European LeukemiaNet-defined Adverse karyotype (HR 0.71 [95%CI 0.51–0.99]; P = 0.046). Azacitidine-treated patients with -5/5q-, -7/7q-, or 17p abnormalities, or with monosomal or complex karyotypes, had a 31–46% reduced risk of death vs CCR. The most frequent gene mutations were DNMT3A (27%), TET2 (25%), IDH2 (23% [R140, 15%; R172, 8%]), and TP53 (21%). Compared with wild-type, OS was significantly reduced among CCR-treated patients with TP53 or NRAS mutations and azacitidine-treated patients with FLT3 or TET2 mutations. Azacitidine may be a preferred treatment for older patients with AML with Adverse-risk cytogenetics, particularly those with chromosome 5, 7, and/or 17 abnormalities and complex or monosomal karyotypes. The influence of gene mutations in azacitidine-treated patients warrants further study.

Progress of Clinical Application of SNP-A to MDS--Review

Cytogenetic abnormalities get wide attention for its guidance value for prognosis and therapy in myelodysplastic syndrome (MDS) and related malignancies. Cytogenetic analysis is also the key to clarify the molecular pathogenesis of these kinds of diseases. The traditional karyotyping technique including metaphase cytogenetic (MC) karyotype analysis and immune fluorescence in situ hybridization (FISH) can detect the chromosomal abnormalities to some degree while the positive rate detected by the techniques is low due to the low resolution, dependence on metaphase dividing cells or the limitation of specific sites on the chromosomes, respectively. Although array comparative genomic hybridization (aCGH) makes up for some deficiencies of the techniques above, only copy number variations (CNVs) could be detected by aCGH. Recently, single nucleotide polymorphasim array (SNP-A) are employed to detect chromosomal CNVs and uniparental disomies (UPDs) which are significant for illumination of the pathogenetics and prognosis of MDS. Based on the detection principle and characteristics of SNP-A, this article reviews the clinical application and prospect of the technique in aspect of the detection characteristic of SNP-A, the relationship between cryptic aberrations and MDS related aspects including the pathogenic genes, phenotypes, prognosis, stratification system and self control test.

Establishment and Characterization of the Novel High-Grade Serous Ovarian Cancer Cell Line OVPA8.

High-grade serous ovarian carcinoma (HGSOC) is the most frequent histological type of ovarian cancer and the one with worst prognosis. Unfortunately, the majority of established ovarian cancer cell lines which are used in the research have unclear histological origin and probably do not represent HGSOC. Thus, new and reliable models of HGSOC are needed. Ascitic fluid from a patient with recurrent HGSOC was used to establish a stable cancer cell line. Cells were characterized by cytogenetic karyotyping and short tandem repeat (STR) profiling. New generation sequencing was applied to test for hot-spot mutations in 50 cancer-associated genes and fluorescence in situ hybridization (FISH) analysis was used to check for TP53 status. Cells were analyzed for expression of several marker genes/proteins by reverse-transcription polymerase chain reaction (RT-PCR), fluorescence-activated cell sorting (FACS), and immunocytochemistry (ICC). Functional tests were performed to compare OVPA8 cells with five commercially available and frequently used ovarian cancer cell lines: SKOV3, A2780, OVCAR3, ES2, and OAW42. Our newly-established OVPA8 cell line shows morphologic and genetic features consistent with HGSOC, such as epithelial morphology, multiple chromosomal aberrations, TP53 mutation, BRCA1 mutation, and loss of one copy of BRCA2. The OVPA8 line has a stable STR profile. Cells are positive for EpCAM, CK19, and CD44; they have relatively low plating efficiency/ability to form spheroids, a low migration rate, and intermediate invasiveness in matrigel, as compared to other ovarian cancer lines. OVPA8 is sensitive to paclitaxel and resistant to cisplatin. We also tested two FGFR inhibitors; OVPA8 cells were resistant to AZD4547 (AstraZeneca, London, UK), but sensitive to the new inhibitor CPL304-110-01 (Celon Pharma, Łomianki/Kiełpin, Poland). We have established and characterized a novel cell line, OVPA8, which can be a valuable preclinical model for studies on high-grade serous ovarian cancer.

Does Solidago litoralis (Asteraceae) merit specific rank? Insights from cytogenetic, molecular and ecological data

Solidago litoralis (Asteraceae) is a psammophile plant endemic to the northern coasts of Tuscany, Italy. During time, different authors have considered it either as a separate species, subspecies or variety of the European S. virgaurea, but few studies of experimental taxonomy have been investigating the relationship between these two taxa. Aim of this study is to compare S. litoralis and S. virgaurea from different points of view: cytogenetic (karyotype analysis, localization of rDNA loci and genome size estimation), molecular (using two plastidial molecular markers), and ecological (by comparing functional characters). A difference in ecological responses to habitats is confirmed, even though S. virgaurea is potentially capable of assuming ecological strategies similar to those of S. litoralis. Despite this, cytogenetic and molecular analyses failed to reveal any significant difference supporting a specific distinction of S. litoralis. The latter taxon is here hypothesized as being an ecotype at the initial steps of a speciation event, better recognized at subspecific level (S. virgaurea subsp. litoralis).

Identification and comparison of individual chromosomes of three accessions of Hordeum chilense, Hordeum vulgare, and Triticum aestivum by FISH.

Karyotypes of three accessions of Hordeum chilense (H1, H16, and H7), Hordeum vulgare, and Triticum aestivum were characterized by physical mapping of several repetitive sequences. A total of 14 repetitive sequences were used as probes for fluorescence in situ hybridization (FISH) with the aim of identifying inter- and intraspecies polymorphisms. The (AG)12 and 4P6 probes only produced hybridization signals in wheat, the BAC7 probe only hybridized to the centromeric region of H. vulgare, and the pSc119.2 probe hybridized to both wheat and H. chilense, but not to H. vulgare. The remaining repetitive sequences used in this study produced a hybridization signal in all the genotypes. Probes pAs1, pTa-535, pTa71, CCS1, and CRW were much conserved, showing no significant polymorphism among the genotypes studied. Probes GAA, (AAC)5, (CTA)5, HvT01, and pTa794 produced the most different hybridization pattern. We identified large polymorphisms in the three accessions of H. chilense studied, supporting the proposal of the existence of different groups inside species of H. chilense. The set of probes described in this work allowed the identification of every single chromosome in all three species, providing a complete cytogenetic karyotype of H. chilense, H. vulgare, and T. aestivum chromosomes, which could be useful in wheat and tritordeum breeding programs.

Integrated Karyotyping of Woodland Strawberry (Fragaria vesca) with Oligopaint FISH Probes

Chromosome identification is critical for many aspects of cytogenetic research. However, for Fragaria vesca, definite identification of individual chromosomes is almost impossible because of their small size and high similarity. Here, we demonstrate that bulked oligonucleotide (oligo) probes can be used as chromosome-specific DNA markers for chromosome identification in F. vesca. Oligos specific to entire pseudochromosomes in the draft genome of F. vesca were identified and synthesized as libraries. In all, we synthesized 6 oligo libraries corresponding to 6 pseudochromosomes of F. vesca. These libraries were amplified and labeled as probes for fluorescence in situ hybridization (FISH). Two rounds of multicolor FISH analysis were sequentially conducted on the same metaphase cells with each round including 3 probe libraries, which permitted simultaneous identification of all chromosomes of F. vesca. Moreover, 45S and 5S rDNA were mapped to chromosomes 1, 2, and 7, respectively. A karyotype of metaphase chromosomes was constructed, representing the first FISH-based molecular cytogenetic karyotype of F. vesca. Our study can serve as a basis for future comparative cytogenetic research through cross-species chromosome painting using bulked oligo probes and will facilitate the application of breeding technologies that rely on the identification of chromosomes in the genus Fragaria.

Novel Structural Abnormalities Involving Chromosomes 1, 17 and 2 Identified by Fluorescence In Situ Hybridization (FISH) and/or Cytogenetic Karyotyping in Kelly and SH-SY5Y Human Neuroblastoma Cell Lines, Respectively

Abstract: MYCN amplification and 1p36 deletion are important poor prognostic factors in neuroblastoma. 1p36 deletion and unbalanced translocations involving chromosomes 1, 17 and 2p were often reported in neuroblastoma cell lines. We aimed to investigate novel chromosomal abnormalities that are likely to affect neuroblastoma progression in Kelly and SH-SY5Y cell lines. Therefore, we analyzed the metaphase chromosomes using fluorescence in situ hybridization (FISH) method with probes specific to the chromosome bands 2p24 and 1p36 in SH-SY5Y and Kelly, respectively. Moreover, the rearrangements of chromosomes 1 and 17 from Kelly were re-examined by cytogenetic karyotyping. FISH analysis shows duplication of chromosome 2p24 on the long arm of a partner chromosome resulted from an unbalanced translocation der(?9)t(2;?9)(p24;q?34) in SH-SY5Y, suggesting that duplication of 2p24 locus containing MYCN gene may contribute to triggering MYCN amplification in neuroblastoma. On the other hand, FISH and karyotype analyses reveal three copies of chromosome 1 that consist of an intact chromosome 1, an extra derivative chromosome 1 with terminal and interstitial deletions (:p32→q25::q41→qter), and another including only interstitial deletion (pter→q25::q41→qter), leading to monosomy of the long arm segment 1q25-q41 in Kelly. These results suggest that chromosome 1q25-q41 may contain one or more tumour suppressor genes important for neuroblastoma progression. Together, FISH analysis shows that an additional 1p36 locus in Kelly is translocated to the short arm of extra chromosome 17, where the p53 tumour suppressor gene is located. Consequently, these novel structural abnormalities involving chromosomes 1, 17 and 2 could be contributed to the tumourigenicity of neuroblastoma cells.

Preferential selection and transfer of euploid noncarrier embryos in preimplantation genetic diagnosis cycles for reciprocal translocations

To develop and validate a new strategy to distinguish between balanced/euploid carrier and noncarrier embryos in preimplantation genetic diagnosis (PGD) cycles for reciprocal translocations and to successfully achieve a live birth after selective transfer of a noncarrier embryo. Retrospective and prospective study. Invitro fertilization (IVF) units. Eleven patients undergoing mate pair sequencing for identification of translocation breakpoints, followed by clinical PGD cycles. Embryo biopsy with 24-chromosome testing to determine carrier status of balanced/euploid embryos. Definition of translocation breakpoints and polymerase chain reaction (PCR) diagnostic primers, correct diagnosis of euploid embryos for carrier status, and a live birth with a normal karyotype after transfer of a noncarrier embryo. In 9 of 11 patients (82%), translocation breakpoints were successfully identified. In four patients with a term PGD pregnancy established with a balanced/euploid embryo of unknown carrier status, the correct carrier status was retrospectively determined, matching with the cytogenetic karyotype of the resulting newborns. In a prospective PGD cycle undertaken by a patient with a 46,XY,t(7;14)(q22;q24.3) translocation, the four balanced/euploid embryos identified comprised three carriers and one noncarrier. Transfer of the noncarrier embryo resulted in birth of a healthy girl who was subsequently confirmed with a normal 46,XX karyotype. The combination of mate pair sequencing and PCR breakpoint analysis of balanced reciprocal translocation derivatives is a novel, reliable, and accurate strategy for distinguishing between carrier and noncarrier balanced/euploid embryos. The method has potential application in clinical PGD cycles for patients with reciprocal translocations or other structural rearrangements.

A rare case of three-way complex variant translocation in chronic myeloid leukemia t(6;9;22)(p21;q34;q11): A case report.

Philadelphia (Ph)-positive chromosome or Ph translocation has been recognized in 90-95 chronic myeloid leukemia (CML) cases worldwide. However, only 5-8% CML patients show complex variant translocations. In the present study, hematological tests for a 47-year-old female CML patient were performed to determine the hemoglobin, platelets and total leukocyte values. A FISH test was carried out to recognize the BCR/ABL gene fusion, and a cytogenetic analysis was performed. The hematological results showed an increase in WBC (414000/mm3) and a decrease in hemoglobin (8.9 mg/dl), indicating the anemic condition in the CML patient. Furthermore, cytogenetic karyotyping results showed 46,XX,t(6;9;22)(p21;q34;q11) and positive for Ph chromosome. In conclusion, in the present study, we report a rare three-way complex variant translocation in a CML patient.

Abstract 994: Diagnosing leukemia structural variations via a novel genome mapping approach

Abstract Identification of structural variants (SVs) is critical to the prognosis of AML. SVs are not readily identified by next generation sequencing methods and are currently diagnosed by cytogenetics, an intrinsically low resolution tool. We have adapted a new method for determining large SVs as a clinical tool for evaluating AML and other cancer genomes. The Bionano Irys images in microfluidic nanochannels large (&amp;gt;150 Kb) segments of genomic DNA that have been bar coded by site specific nicking and local incorporation of fluorescent label. The complement of individual molecules can be assembled through matching bar codes to create a de novo map of the genome from which, by matching to a reference genome, identifies and catalogs structural variants at resolution of ca. 1 Kb. With this technology, we characterized structural variants signatures in breast cancer, renal clear cell carcinoma, and CML cell lines. Structural variants were then validated via PCR/Sanger sequencing, fluorescent in situ hybridization and whole genome sequencing. We identified a thorough compendium of consistent false positive rearrangements identified by this technology, most of which reflect errors in the reference human genome assembly. We then applied this methodology to ten AML patient samples, seven from archived blood samples and three directly obtained from newly diagnosed patients. Karyotyping on one archived sample identified a t(6:9) translocation in the majority of cells. We identified that rearrangement at a much higher resolution than cytogenetics, which allowed us to map the breakpoint via PCR/Sanger sequencing to nucleotide resolution and confirm a fusion of NUP214 to DEK, a known oncogene. We also discovered the reciprocal t(9:6) translocation, which had not been identified by cytogenetics. In the six other cases from archived samples, we have confirmed the translocation identified by cytogenetic karyotyping and in several cases found additional rearrangements. Moreover, in the first pediatric case, which we completed within five days of obtaining the sample, we have identified four distinct translocations, all of which have been associated previously with AML. Overall, this is technology has the ability to quickly and accurately identify SVs in cancer cell lines and AML patient samples with higher resolution than cytogenetics, demonstrating its clinical usefulness in the diagnosis and subsequent treatment of leukemia. Citation Format: Christopher Pool, Jie Xu, Darrin Bann, David Goldenberg, Arati Sharma, David Claxton, Feng Yue, James Broach. Diagnosing leukemia structural variations via a novel genome mapping approach [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 994. doi:10.1158/1538-7445.AM2017-994

Abstract 1766: Distinct pattern of alterations in tp53 mutated and wild type acute myeloid leukemia (AML) patients

Abstract Background: Mutations in TP53 gene predict a poor prognosis in patients with AML. The reported TP53 mutation rate in AML is low (2.1%) by contrast AML with a complex karyotype (CK) is higher (69-78%) and have a poor outcome. Quite common is to found paired TP53 mutation together and a segmental 17p deletion. Aims: To investigate the frequency, the types of mutations, the associated cytogenetic, the correlation with known molecular alterations and the prognostic role TP53 mutations in adult AML pts. Moreover we would identify genes/pathways that are mainly affected in the mutated TP53 group compared to the wt one. Patients and Methods: 258 adult AML pts with miscellaneous cytogenetic abnormalities and normal karyotype were examined in our Institution for TP53 mutations using several methods, including Sanger sequencing, NGS and HiSeq2000 platform and were correlated with cytogenetic analysis. 124/258 samples were genotyped with SNP arrays (Affymetrix, 3 250K, 43 SNP 6.0, 78 CytoScan HD). Copy Number Alterations (CNAs) analyses were performed using Chromosome Analysis Suite (Affymetrix) and Nexus Copy Number (BioDiscovery) software. Results: Mutation analysis detected TP53 mutations on 39 patients with 48 different types of mutations (32 deleterious point mutations; 4 deletions); nine pts have 2 mutations. We found 34/48 (70%) missense mutations, 5 mutations in the splice sites, 4 deletions ,2 intronic and and 3 others mutations. The mutation rate is of 15.1%. Mostly (28/39) of the TP53 mutated pts (71.8%) had CK while only 11/39 (28.2%) mutated pts have “no CK” (P&amp;gt;0.0001). Alterations of TP53 were significantly associated with poor outcome (OS and EFS p&amp;lt;0.0001). To take advantage of different methodological characteristics, on TP53 locus, we matched two software and cytogenetic analysis results. We identify 16 mutated pts that were also deleted and one pt that presented only a deletion. Therefore 50% of mutated pts present a concomitant deletion. OS of TP53 mutated and deleted pts is statistically inferior respect to muted pts (p&amp;lt;0.0061). Comparing 32 TP53 mutated and 92 TP53 wt pts CNAs results that: a) chromosomes significantly alterated are 5q, 17p, 12p, 16q, 22q13.33 and 7q; b) over 900 genes are differentially involved (all in Loss); c) and that pathway categories mainly enriched are Signal Transduction, Metabolism, Immune System, Transmembrane transport of small molecules, Gene expression, Cell Cycle. Conclusions: Our data demonstrated that TP53 mutations occur in 15.1% of AML with a higher frequency in the subgroup of CK-AML (p&amp;lt;0.0001). They predicted to be deleterious and significantly correlated with worse prognosis especially if TP53 is both mutated and also deleted. For these reasons, TP53 mutation/deletion screening should be recommended. Different pattern of alterations in mutated and wt groups have to be deeper investigated. ELN, AIL, AIRC, progetto Regione-Università 2010-12 (L. Bolondi), FP7 NGS-PTL project. Citation Format: Anna Ferrari, Eugenio Fonzi, Maria Chiara Fontana, Andrea Ghelli Luserna Di Rorà, Marco Manfrini, Antonella Padella, Carmen Baldazzi, Cristina Papayannidis, Giovanni Marconi, Stefania Paolini, Viviana Guadagnuolo, Margherita Perricone, Valentina Robustelli, Enrica Imbrogno, Eugenia Franchini, Claudia Venturi, Elisa Zuffa, Maria Chiara Abbenante, Giorgia Simonetti, Nicoletta Testoni, Emanuela Ottaviani, Martinelli Giovanni. Distinct pattern of alterations in tp53 mutated and wild type acute myeloid leukemia (AML) patients [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2017; 2017 Apr 1-5; Washington, DC. Philadelphia (PA): AACR; Cancer Res 2017;77(13 Suppl):Abstract nr 1766. doi:10.1158/1538-7445.AM2017-1766