Acquisition Of Chromosomal Abnormalities Research Articles

Targeting histone methyltransferase and demethylase in acute myeloid leukemia therapy.

Acute myeloid leukemia (AML) is a clonal disorder of myeloid progenitors characterized by the acquisition of chromosomal abnormalities, somatic mutations, and epigenetic changes that determine a consistent degree of biological and clinical heterogeneity. Advances in genomic technologies have increasingly shown the complexity and heterogeneity of genetic and epigenetic alterations in AML. Among the genetic alterations occurring in AML, frequent are the genetic alterations at the level of various genes involved in the epigenetic control of the DNA methylome and histone methylome. In fact, genes involved in DNA demethylation (such as DNMT3A, TET2, IDH1, and IDH2) or histone methylation and demethylation (EZH2, MLL, DOT1L) are frequently mutated in primary and secondary AML. Furthermore, some histone demethylases, such as LSD1, are frequently overexpressed in AML. These observations have strongly supported a major role of dysregulated epigenetic regulatory processes in leukemia onset and development. This conclusion was further supported by the observation that mutations in genes encoding epigenetic modifiers, such as DMT3A, ASXL1, TET2, IDH1, and IDH2, are usually acquired early and are present in the founding leukemic clone. These observations have contributed to development of the idea that targeting epigenetic abnormalities could represent a potentially promising strategy for the development of innovative treatments of AML. In this review, we analyze those proteins and their inhibitors that have already reached the first stages of clinical trials in AML, namely the histone methyltransferase DOT1L, the demethylase LSD1, and the MLL-interacting protein menin.

Ovine induced pluripotent stem cells are resistant to reprogramming after nuclear transfer.

Induced pluripotent stem cells (iPSCs) share similar characteristics of indefinite in vitro growth with embryonic stem cells (ESCs) and may therefore serve as a useful tool for the targeted genetic modification of farm animals via nuclear transfer (NT). Derivation of stable ESC lines from farm animals has not been possible, therefore, it is important to determine whether iPSCs can be used as substitutes for ESCs in generating genetically modified cloned farm animals. We generated ovine iPSCs by conventional retroviral transduction using the four Yamanaka factors. These cells were basic fibroblast growth factor (bFGF)- and activin A-dependent, showed persistent expression of the transgenes, acquired chromosomal abnormalities, and failed to activate endogenous NANOG. Nonetheless, iPSCs could differentiate into the three somatic germ layers in vitro. Because cloning of farm animals is best achieved with diploid cells (G1/G0), we synchronized the iPSCs in G1 prior to NT. Despite the cell cycle synchronization, preimplantation development of iPSC-NT embryos was lower than with somatic cells (2% vs. 10% blastocysts, p<0.01). Furthermore, analysis of the blastocysts produced demonstrated persistent expression of the transgenes, aberrant expression of endogenous SOX2, and a failure to activate NANOG consistently. In contrast, gene expression in blastocysts produced with the parental fetal fibroblasts was similar to those generated by in vitro fertilization. Taken together, our data suggest that the persistent expression of the exogenous factors and the acquisition of chromosomal abnormalities are incompatible with normal development of NT embryos produced with iPSCs.

The Genomic Landscape of Relapsed B-Cell Acute Lymphoblastic Leukaemia (B-ALL) in Adolescents and Adults

Despite significant advancements in the treatment of paediatric B-cell acute lymphoblastic leukaemia (B-ALL), ALL remains one of the most challenging adult malignancies. Outcome of adult B-ALL is poor with only 40% 5-year event-free survival compared to >80% in children. B-ALL is characterised by the acquisition of chromosomal abnormalities and many are strong predictors of outcome. The difference in the prevalence of cytogenetic subtypes and specific genomic abnormalities observed between adult and childhood ALL suggests a difference in tumour biology that may contribute to the differences in patient outcome. Detailed analysis of the paediatric B-ALL genome have revealed a plethora of abnormalities targeting key pathways. Although specific alterations have been investigated in adult and adolescent B-ALL, studies of the genomic landscape remain scarce. In this study we set-out to define the genomic landscape of adolescent and adult relapsed B-ALL. Genomic backtracking analysis of sequential diagnostic and relapse samples revealed known and novel abnormalities that may play a role in chemoresistance and disease progression in these tumours.DNA was isolated from the diagnostic and relapse samples from 12 adolescents/adult patients (5 female and 7 male) diagnosed with B-ALL. Eight of the 12 patients had remission samples available. Known cytogenetic abnormalities were detected in 7 patients: high hyperdiploid, t(1;19), t(8;14) and t(4;11) rearrangements. Two cases were positive for the BCR-ABLfusion gene. The mean age at diagnosis was 36.8 years (range 16-59 years) of which 10 relapsed early, within 2 years of initial diagnosis. DNA for the 12 diagnostic, 12 relapse and 8 remission sample were hybridised to the Affymetrix SNP6.0 array to determine copy number abnormalities (CNAs). The mutational landscape was captured for 4 cases using the Agilent SureSelect Human All Exon V4+UTR kit and sequenced to depths of 200X.The incidences of the most prevalent abnormalities in paediatric B-ALL were determined in each adult/adolescent sample: CDKN2A/B 88% (21/24), IKZF1 20% (5/24), PAX5 8% (2/24), ETV6 0% (0/24), RB1 8% (2/24), BTG1 8% (2/24) and EBF1 17% (4/24). Deletions of CDKN2A/B were detected in all but one patient. In 9 cases the abnormality was seen at both diagnosis and relapse and one case had a de novo deletion at relapse. A further case had a sub-clone harbouring CDKN2A/B deletion at diagnosis that emerged as the dominant clone at relapse. Deletion of CDKN2A/Bhas been associated with poor overall survival and has been reported at high incidence in relapsed adult BCR-ABL1-ALL, but the association with prognosis and relapse in other subtypes has not been confirmed.Genomic backtracking analysis of the matched diagnostic and relapse samples identified, on average, 36 somatic mutations at relapse that were either not detected or were only detectable in a sub-clone at diagnosis. An average of 0.05 mutations per Mb were computationally predicted to be damaging to the function of the protein. Novel de novo mutations seen at relapse were identified in cancer-related genes: FAT4, CDCA7 and PVRL4. Sequencing at depth of >200X demonstrates the ability to detect mutations in the resistant clone which could be involved in disease progression. Mutations in the ATP-binding cassette transporter gene, ABCC9, were identified in a sub-clone at diagnosis at a variant frequency of 5% (13/268 reads) and at 43% (113/260) in the relapse sample. ABCC9is involved in drug resistance suggesting a potential role in chremoresistance in this patient.In conclusion, in-depth genomic analysis and whole-exome sequencing of matched diagnostic and relapse samples in adult/adolescent B-ALL has identified known and novel genomic abnormalities. Deletion of CDKN2A/B was prevalent in 11 of the 12 cases confirming the importance of this region in relapsed B-ALL. We have identified novel mutations in genes associated with chemoresistance and tumorigenesis: ABCC9, FAT4, CDCA7 and PVRL4. Our study provides the most comprehensive genetic portrait of adult relapsed B-ALL to date and is a significant step to defining the genetic causes of disease progression and chemoresistance. DisclosuresNo relevant conflicts of interest to declare.

Clinical Impact of Complex Karyotype, Monosomal Karyotype and Acquisition of Chromosomal Abnormalities in Patients with Myelodysplastic Syndromes

ObjectivesDespite azacitidine prolonging the survival of patients (pts) with myelodysplastic syndromes (MDS), the overall survival (OS) of those with complex karyotype (CK) or monosomal karyotype (MK) remains dismal, and optimal treatments for these pts have not been established. Furthermore, the prognostic impact of acquisition of additional cytogenetic abnormalities (ACA) during the follow-up periods is poorly understood. In this retrospective study, we aimed to evaluate the prognostic impact of CK, MK, and ACA in MDS pts, and determine optimal treatments.MethodsWe collected clinical data on consecutive adult MDS pts who first visited Kobe City Medical Center General Hospital or Kyoto University Hospital between January 2004 and April 2014. Patients were divided into groups based on the number of chromosomal abnormalities (abns) and presence or absence of MK. ACA was defined as any gain in the number of chromosomal abns before treatments. Survival analysis was adjusted using the following variables; sex, age, IPSS-R parameters and MK. A Cox regression model was applied to the results. ACA was fit into the model as a time-dependent covariate.ResultsA total of 299 pts with MDS were identified, five of which were excluded from the study due to a lack of chromosomal information. Median follow-up time was 24.5 months (M). Pts’ characteristics, treatments and incidence of ACA are summarized in Table 1. Among 47 CK pts, 34 (72%) carried MK. Survival analysis revealed that presence of 4 or more chromosomal abns was significantly associated with increased mortality. Among pts with 2–3 chromosomal abns, the presence of MK was a poor prognostic factor, although it had no significant impact on survival in pts with 4 or more abns (Figure 1). Median survival times of pts with 2–3-abns without MK, with 2–3-abns together with MK, and 4 or more abns were 39 M, 12 M (HR 6.6, 95% confident interval, [2.6-17], p < 0.001), and 9 M(HR 5.4, [3.1–9.3], p < 0.001) respectively. Regarding ACA, changes from normal karyotype (NK) to 1 abn (N=15) had no effect on mortality, but changes from NK to 2 or more abns (N=10) led to a significant increase in mortality (HR 9.2 [3.7–24], p < 0.001). Further, changes from 0–3 chromosomal abns without MK to either MK or 4 or more abns were associated with higher mortality (HR 2.7 [1.0–7.1], p = 0.045). Among pts with 2–3-chromosomal abns with MK or those with 4 or more abns at diagnosis (N=45) or during follow-up (N=10), 20 (36%) underwent allogeneic stem cell transplantation (allo-SCT) and only six survived beyond 24 M. Five of these received allo-SCT and were alive at the last follow-up. The other was treated with azacitidine and lived for 33 M.ConclusionThis study revealed that MK or 4 or more chromosomal abns is significantly associated with high mortality. Among NK pts, acquisition of 2 or more abns was associated with a poor outcome. Allo-SCT is the only strategy to increase the chance of long-term survival for pts with MK or 4 or more chromosomal abns, although only a few survivors were identified even after allo-SCT. More data are needed to identify the optimal therapeutic options for this subgroup.Table 1Clinical parameters and treatments received by patientsNumber of abnormalities<3 (N=247) (%)3 (N=10) (%)4<= (N=37) (%)p-valueSex (male/female) (N=195/99)170(69)/77(31)4(40)/6(60)21(57)/16(43)0.04Age =<65 (N=158) >65 (N=136)130(53) 117(47)7(70) 3(30)21(57) 16(43)0.89Bone marrow blast% <=2 (N=123) >2-<5 (N=45) >=5-<=10 (N=50) >10 (N=74) Missing (N=2)112(45) 35(14) 39(16) 59(24) 2(0.81)4(40) 3(30) 2(20) 1(10) 07(19) 7(19) 9(24) 14(38) 00.06Hemoglobin (g/dL) >=10 (N=91) >=8-<10 (N=93) <8 (N=106) Missing (N=4)83(34) 72(29) 88(36) 4(1.6)3(30) 5(50) 2(20) 05(14) 16(43) 16(43) 00.076Platelets (103/ƒÊL) >=100 (N=118) >50-<100 (N=82) <50 (N=90) Missing (N=4)100(40) 68(28) 75(30) 4(1.6)6(60) 1(10) 3(30) 0(0)12(32) 13(35) 12(32) 00.50Neutrophils (/ƒÊL) >=800 (N=183) <800 (N=106) Missing (N=5)160(65) 82(33) 5(2.0)5(50) 5(50) 018(49) 19(51) 00.078Monosomal karyotype (N=39)5(2)3(30)31(79)<0.001ACA (N=37)30(12)1(10)6(16)0.7Initial treatments CC (N=92) azacitidine (±CC) (N=63) Chemo-naïve allo-SCT (N=37) Palliative CT/BSC (N=102)78(32) 43(17) 31(13) 95(38)1(10) 3(30) 3(30) 3(30)13(35) 17(46) 3(8) 4(11)<0.001Allo-SCT (total)79(32)7(70)13(35)0.77 [Display omitted] DisclosuresNo relevant conflicts of interest to declare.

Chromosome instability in mouse embryonic stem cells.

Embryonic Stem Cells (ESCs) are expected to show a stable euploid karyotype, but in the last decade (sub)chromosomal aberrations have been systematically described in these cell lines when maintained in vitro. Culture conditions and long-term culture have been traditionally proposed as possible factors involved in the acquisition of chromosomal abnormalities. Thus, we analyzed the chromosome constitution, the undifferentiated state and the functional pluripotency of three different mouse ESCs grown under the same culture conditions. Two cell lines were unstable from early passages, whereas the third one retained its chromosome integrity after long-term culture despite using enzymatic methods for cell disaggregation. Trisomy 8 and 11 were clonally selected in both unstable cell lines, which also showed a higher growth rate than our normal cell line and suffered morphological changes in colony shape with increasing passage number. Regardless of the length of culture or the chromosome instability, all cell lines preserved their differentiation potential. These results confirm that double trisomy 8 and 11 confers a growth advantage to the abnormal cells, but not at the expense of cell differentiation. The presence of chromosome instability, widely related to tumor development and cancer disease, highlights the risk of using pluripotent cells in regenerative medicine.

Aneuploidy in pluripotent stem cells and implications for cancerous transformation.

Owing to a unique set of attributes, human pluripotent stem cells (hPSCs) have emerged as a promising cell source for regenerative medicine, disease modeling and drug discovery. Assurance of genetic stability over long term maintenance of hPSCs is pivotal in this endeavor, but hPSCs can adapt to life in culture by acquiring non-random genetic changes that render them more robust and easier to grow. In separate studies between 12.5% and 34% of hPSC lines were found to acquire chromosome abnormalities over time, with the incidence increasing with passage number. The predominant genetic changes found in hPSC lines involve changes in chromosome number and structure (particularly of chromosomes 1, 12, 17 and 20), reminiscent of the changes observed in cancer cells. In this review, we summarize current knowledge on the causes and consequences of aneuploidy in hPSCs and highlight the potential links with genetic changes observed in human cancers and early embryos. We point to the need for comprehensive characterization of mechanisms underpinning both the acquisition of chromosomal abnormalities and selection pressures, which allow mutations to persist in hPSC cultures. Elucidation of these mechanisms will help to design culture conditions that minimize the appearance of aneuploid hPSCs. Moreover, aneuploidy in hPSCs may provide a unique platform to analyse the driving forces behind the genome evolution that may eventually lead to cancerous transformation.

Hypoxia-induced reactive oxygen species cause chromosomal abnormalities in endothelial cells in the tumor microenvironment.

There is much evidence that hypoxia in the tumor microenvironment enhances tumor progression. In an earlier study, we reported abnormal phenotypes of tumor-associated endothelial cells such as those resistant to chemotherapy and chromosomal instability. Here we investigated the role of hypoxia in the acquisition of chromosomal abnormalities in endothelial cells. Tumor-associated endothelial cells isolated from human tumor xenografts showed chromosomal abnormalities, >30% of which were aneuploidy. Aneuploidy of the tumor-associated endothelial cells was also shown by simultaneous in-situ hybridization for chromosome 17 and by immunohistochemistry with anti-CD31 antibody for endothelial staining. The aneuploid cells were surrounded by a pimonidazole-positive area, indicating hypoxia. Human microvascular endothelial cells expressed hypoxia-inducible factor 1 and vascular endothelial growth factor A in response to either hypoxia or hypoxia-reoxygenation, and in these conditions, they acquired aneuploidy in 7 days. Induction of aneuploidy was inhibited by either inhibition of vascular endothelial growth factor signaling with vascular endothelial growth factor receptor 2 inhibitor or by inhibition of reactive oxygen species by N-acetyl-L-cysteine. These results indicate that hypoxia induces chromosomal abnormalities in endothelial cells through the induction of reactive oxygen species and excess signaling of vascular endothelial growth factor in the tumor microenvironment.

High-resolution DNA analysis of human embryonic stem cell lines reveals culture-induced copy number changes and loss of heterozygosity

Prolonged culture of human embryonic stem cells (hESCs) can lead to adaptation and the acquisition of chromosomal abnormalities, underscoring the need for rigorous genetic analysis of these cells. Here we report the highest-resolution study of hESCs to date using an Affymetrix SNP 6.0 array containing 906,600 probes for single nucleotide polymorphisms (SNPs) and 946,000 probes for copy number variations (CNVs). Analysis of 17 different hESC lines maintained in different laboratories identified 843 CNVs of 50 kb-3 Mb in size. We identified, on average, 24% of the loss of heterozygosity (LOH) sites and 66% of the CNVs changed in culture between early and late passages of the same lines. Thirty percent of the genes detected within CNV sites had altered expression compared to samples with normal copy number states, of which >44% were functionally linked to cancer. Furthermore, LOH of the q arm of chromosome 16, which has not been observed previously in hESCs, was detected.

Establishment of a leukaemic cell line from a patient with acquisition of chromosomal abnormalities during disease progression in myelodysplastic syndrome

A cell line designated SKM-1 was newly established from leukaemic cells of a 76-year-old Japanese male patient with monoblastic leukaemia following myelodysplastic syndrome (MDS). The cells were obtained from peripheral blood of the patient when he lost multiple point mutations of ras genes with acquisition of chromosomal abnormalities during disease progression in MDS. The cells grew as a single floating cell, and have been continuously growing with the morphological characteristics of immature monoblasts by serial passages during the past 42 months with a doubling time of about 48 h. By cytochemical analysis, the cloned cells were positive for butyrate esterase, but negative for the Epstein-Barr virus associated nuclear antigen. Phenotypic analysis revealed the expression of myelomonocyte specific antigens such as CD4, CD13, CD33 and HLA-DR. Cells from the primary peripheral blood and those from 50 passages of the SKM-1 cell line both possessed no activated ras genes but showed karyotype abnormalities with 46,XY, del(9)(q13;q22), der(17) t(17;?)(p13;?). The SKM-1 cells have two mutations in p53 gene and overexpress the p53 products. This cell line may contribute to a better understanding of molecular mechanisms in the progression from MDS to myelogenous leukaemia.