Specific Chromosomal Abnormalities Research Articles

Hypoplastic left heart syndrome and other left heart disease: evolution of understanding from population-based analysis to molecular biology and back again – a brief overview

Congenital cardiac disease is the most common birth defect, occurring in approximately 1 in 1000 live births. Congenital cardiac defects have associations, whether with gender, race, or specific chromosomal abnormalities, potentially allowing grouping of defects to be studied in an effort to develop an understanding of aetiological factors. The Baltimore-Washington Infant Study provides full ascertainment of a population of infants with congenital cardiac disease born in a defined geographic region. The fundamental hypotheses generated at the inception of the Baltimore-Washington Infant Study included the central idea that the outcome of birth, including the development of congenital cardiac malformations, was influenced by environmental factors and their route of introduction into a genetically susceptible host. Evidence exists that supports the concept that both genetic and environmental factors contribute to the development of diseases of the left heart.

Impact of gene expression profiling-based risk stratification in patients with myeloma receiving initial therapy with lenalidomide and dexamethasone

AbstractDetection of specific chromosomal abnormalities by FISH and metaphase cytogenetics allows risk stratification in multiple myeloma; however, gene expression profiling (GEP) based signatures may enable more specific risk categorization. We examined the utility of 2 GEP-based risk stratification systems among patients undergoing initial therapy with lenalidomide in the context of a phase 3 trial. Among 45 patients studied at baseline, 7 (16%) and 10 (22%), respectively, were high-risk using the GEP70 and GEP15 signatures. The median overall survival for the GEP70 high-risk group was 19 months versus not reached for the rest (hazard ratio = 14.1). Although the medians were not reached, the GEP15 also predicted a poor outcome among the high-risk patients. The C-statistic for the GEP70, GEP15, and FISH based risk stratification systems was 0.74, 0.7, and 0.7, respectively. Here we demonstrate the prognostic value for GEP risk stratification in a group of patients primarily treated with novel agents. This trial was registered at www.clinicaltrials.gov as #NCT00098475.

Immunophenotype and cytogenetic characteristics in the relationship between birth weight and childhood leukemia

High birth weight increases the risk of childhood acute lymphoid leukemia (ALL) through unknown mechanisms. Whether this risk is specific to ALL subtypes is unknown, and low case numbers have prevented investigation of the rarer leukemias. Here we address these associations using a large population-based dataset. Using the National Registry of Childhood Tumors, birth weights of 7,826 leukemia cases, defined by immunophenotype and cytogenetic subgroup, were compared with those of 10,785 controls born in England and Wales between 1980 and 2007. The risk for overall leukemia increases 7% with each 0.5 kg increase in birth weight (OR 1.07, 95%CI 1.04-1.10). This risk is limited to the lymphoid leukemias (OR 1.08, 95%CI 1.05-1.12) diagnosed between 1 and 9 years of age. Analysis by cytogenetic feature reveals that there appears to be association with specific chromosomal abnormality: the risk of tumors with high hyperdiploid karyotypes increases 12% per 0.5 kg increase in birth weight (OR 1.12, 95%CI 1.05-1.20), and t(1;19) tumors show an increased risk of 41% per 0.5 kg increase (OR 1.41, 95%CI 1.09-1.84). The risk of acute myeloid leukemia is elevated in high and low birth weight babies. There is no significant risk relationship to other leukemias or myeloproliferative diseases. Birth weight is a risk factor for ALL and AML. Other subtypes of the disease are not significantly affected. There appears to be association with specific chromosomal abnormality, which may aid our understanding of the development of childhood leukemia in utero.

Cytogenetic abnormalities in myelodysplastic syndrome: an overview

The myelodysplastic syndromes (MDS) are clonal disorders of haematopoietic stem cells characterized by ineffective haematopoiesis leading to blood cytopenias and by high incidence of progression to acute myeloid leukaemia (AML). These disorders generally arise de novo, but may also occur years after exposure to mutagenic chemotherapy. Clonal cytogenetic abnormalities are detected in about 30–50% de novo cases, whereas more than 80% of therapy-related forms harbour such markers. Although in the Western countries, MDS cases are mainly reported in the elderly population and rarely in the paediatric age group; this disease is increasingly seen in young adults in India. Cytogenetic study plays an important role in the diagnosis and is useful for prediction of individual prognosis using the international prognostic scoring system. Specific chromosomal abnormalities, such as −5/5q-, −7/7q-, and complex abnormalities, play an important role in the development of new therapeutic options and clinical management of MDS. In this review, we summarize the cytogenetic abnormalities in MDS from various parts of the world.

Annual Research Review: Transgenic mouse models of childhood‐onset psychiatric disorders

Childhood‐onset psychiatric disorders, such as attention deficit hyperactivity disorder (ADHD), autism spectrum disorder (ASD), mood disorders, obsessive compulsive spectrum disorders (OCSD), and schizophrenia (SZ), affect many school‐age children, leading to a lower quality of life, including difficulties in school and personal relationships that persist into adulthood. Currently, the causes of these psychiatric disorders are poorly understood, resulting in difficulty diagnosing affected children, and insufficient treatment options. Family and twin studies implicate a genetic contribution for ADHD, ASD, mood disorders, OCSD, and SZ. Identification of candidate genes and chromosomal regions associated with a particular disorder provide targets for directed research, and understanding how these genes influence the disease state will provide valuable insights for improving the diagnosis and treatment of children with psychiatric disorders. Transgenic mouse models are one important approach in the study of human diseases, allowing for the use of a variety of experimental approaches to dissect the contribution of a specific chromosomal or genetic abnormality in human disorders. While it is impossible to model an entire psychiatric disorder in a single mouse model, these models can be extremely valuable in dissecting out the specific role of a gene, pathway, neuron subtype, or brain region in a particular abnormal behavior. In this review we discuss existing transgenic mouse models for childhood‐onset psychiatric disorders. We compare the strength and weakness of various transgenic mouse models proposed for each of the common childhood‐onset psychiatric disorders, and discuss future directions for the study of these disorders using cutting‐edge genetic tools.

Chromosomal abnormalities in renal cell carcinoma variants detected by Urovysion fluorescence in situ hybridization on paraffin-embedded tissue

Urovysion fluorescence in situ hybridization (UVFISH) identifies malignant cells in urine by detecting specific urothelial carcinoma-related chromosomal abnormalities. Some renal carcinomas (RCCs) share overlapping chromosomal aberrations with urothelial carcinoma. Malignant renal cells that are shed in urine can potentially cause a positive UVFISH result. We evaluated UVFISH in RCCs to determine its potential applicability to the diagnosis and grading of RCCs. Paraffin blocks from 39 RCCs (25 clear cell, 9 papillary, 2 chromophobe, and 3 sarcomatoid) and 15 controls (5 renal oncocytomas and 10 urothelial carcinomas) were tested. Of the RCCs, 15 (40%) were UVFISH-positive (9/25 [40%] clear cell, 3/9 [30%] papillary, 1/2 [50%] chromophobe, and 2/3 [67%] sarcomatoid carcinoma) and 24 (60%) were negative. Of the 15 controls, 8 (∼50%) were UVFISH-positive (2/5 [40%] oncocytomas and 6/10 [60%] urothelial carcinomas) and 7 (∼50%) were UVFISH-negative. Polysomy of chromosome 17 showed a statistically significant correlation with RCC subtype, being absent in most of the clear cell RCCs (P = .0096) compared with other RCCs. Polysomy of chromosome 7 was more frequent in high-grade than low-grade RCC (P = .0197) and more likely in high-grade clear cell than low-grade clear cell RCC (P = .0120). In conclusion, we showed that RCC has overlapping chromosomal abnormalities with urothelial carcinoma and can cause a positive UVFISH result. This has implications for the interpretation of Urovysion in patients whose urine contains malignant cells but who have negative cystoscopy and a concomitant renal mass. The chromosomal abnormalities observed in RCC are not distinct from those in urothelial carcinoma; therefore, UVFISH cannot distinguish these tumor types, nor can it type or grade RCC.

Craniosynostosis

Craniosynostosis, defined as the premature fusion of the cranial sutures, presents many challenges in classification and treatment. At least 20% of cases are caused by specific single gene mutations or chromosome abnormalities. This article maps out approaches to clinical assessment of a child presenting with an unusual head shape, and illustrates how genetic analysis can contribute to diagnosis and management.

Acquired genomic copy number aberrations and survival in adult acute myelogenous leukemia

AbstractGenomic aberrations are of predominant importance to the biology and clinical outcome of patients with acute myelogenous leukemia (AML), and conventional karyotype-based risk classifications are routinely used in clinical decision making in AML. One of the known limitations of cytogenetic analysis is the inability to detect genomic abnormalities less than 5 Mb in size, and it is currently unclear whether overcoming this limitation with high-resolution genomic single-nucleotide polymorphism (SNP) array analysis would be clinically relevant. Furthermore, given the heterogeneity of molecular mechanisms/aberrations that underlie the conventional karyotype-based risk classifications, it is likely that further refinements in genomic risk prognostication can be achieved. In this study, we analyzed flow cytometer–sorted, AML blast-derived, and paired, buccal DNA from 114 previously untreated prospectively enrolled AML patients for acquired genomic copy number changes and loss of heterozygosity using Affymetrix SNP 6.0 arrays, and we correlated genomic lesion load and specific chromosomal abnormalities with patient survival. Using multivariate analyses, we found that having ≥ 2 genomic lesions detected through SNP 6.0 array profiling approximately doubles the risk of death when controlling for age- and karyotype-based risk. Finally, we identified an independent negative prognostic impact of p53 mutations, or p53 mutations and 17p-loss of heterozygosity combined on survival in AML.

Various Abnormalities at Chromosome 7 Carry Distinct Biologic and Prognostic Implications In Myelodysplastic/Myeloproliferative Syndromes and Related Marrow Failures

AbstractAbstract 2744Deletions involving chromosome 7 are frequently encountered in myeloid malignancies, mostly imparting a poor prognosis. Single nucleotide polymorphism array (SNP-A)-based cytogenetics has allowed for an improvement in the mapping of disease-related chromosomal regions through identification of microdeletion that define minimally affected areas for the search for pathogenic mutations. In addition, SNP-A facilitated the detection of somatic uniparental disomy (sUPD), which is frequently found in myeloid malignancies. Regions of sUPD are often associated with homozygous mutations present in the affected area, as shown for example for UPD9p and JAK2 or UPD11q and C-CBL mutations. Based on the precise mapping of genomic lesions on chromosome 7, we compared the outcomes of patients characterized by specific chromosomal abnormalities. Among 1163 patients with various myeloid malignancies studied by SNP-A we identified patients with somatic LOH involving chromosome 7 for whom clinical outcome data was available to assess survival. Regions fulfilling the criteria of germ line-encoded copy number variants and regions of homozygosity present in a control cohort (N=1535) were excluded. The rest of newly observed regions were in/excluded based on their absence or presence in the paired non-clonal DNA. In total, we identified 45 patients with monosomy 7, 37 patients with del(7q), and 21 with UPD7q. The CDR for del(7q) and UPD(7q) spanned nt 115221516–126602492 and nt 127,484,450–158821424, respectively. Among patients with monosomy 7, 17 were primary MDS, 22 had various bone marrow failure disorders including juvenile myelomonocytic leukemia (JMML) and clonal disease in the context of aplastic anemia (AA) and Fanconi anemia (FA), whereas UPD7q was predominantly found in MDS/MPD and MPD (N=10), sAML (N=3), and primary MDS (N=7). Patients with del(7q) included primary MDS and sAML. The most common concomitant cytogenetic abnormalities were seen in chromosome 5q and 17p, including frequent UPD17p. Chromosome 5q and 17p lesions were more common in del(7q), identified in 10 and 7 patients respectively, and were represented equivalently among monosomy 7 (N=5 for each) and UPD7q patients (N=3 and 5 respectively). In addition to the large deletions of the long arm of chromosome 7 ([del(7q)] discussed above, using SNP-A-based karyotyping, 18 patients were identified with overlapping microdeletions involving two distinct loci at 7q22.1 (N=5 and 6, respectively), and one at 7q36.1 (N=2). The first 7q22.1 loci was found in 1 patient with MDS and 5 with MPN, while the second loci was identified in 3 patients with chronic myelomonocytic leukemia (CMML), 1 with MPN, and 1 with sAML. The 7q36.1 minimally affected region was found in 2 patients, 1 each with MDS and CMML. In 15 patients non-recurrent microdeletions were found with average size of 799 kb.Clinical parameters were compared for each abnormality and Kaplan-Meier plots were generated and evaluated by log-rank test. Comparison of monosomy 7 and del(7q) did not show statistically different impact on outcome (p=0.26), whereas each was found to carry a significantly poorer prognosis (OS) than UPD7 (p=0.05 and 0.02 respectively). Deletion of 7q36.1, whose common deleted region was consistent with our previous identification of mutation within EZH2, carried an overall poor prognosis while 7q22.1 did not affect survival compared to patients without chromosome 7 abnormalities. These data confirm the known poor outcomes of patients with chromosome 7 abnormalities shared among monosomy 7 as well as del(7q). A slightly better albeit poor prognosis was seen in UPD(7q), suggesting that pattern or inheritance of chromosome 7 abnormalities into the dysplastic clone may affect ultimate leukemogenicity. UPD7q may represent a distinct pathogenesis, likely with distinct gene mutations compared to del(7q) and monosomy 7. Somatic microdeletions in chromosome 7 analysis may be nonpathogenic while those with poor prognosis, such as 7q36.1, may harbor pathogeneic genes. Disclosures:No relevant conflicts of interest to declare.

SNP-Arrays: a Routine Cytogenetic Tool In Chronic Lymphocytic Leukemia ?.

AbstractAbstract 4611 Background:Chronic lymphocytic leukemia (B-CLL) is the most common adult leukemia in Western countries, and is characterized by a highly variable clinical course. Interphase fluorescent in situ hybridization (I-FISH) has been able to identify chromosomal abnormalities in ~80% of B-CLL, including deletions at 13q, 11q, 17p and trisomy 12, which has proven to be prognostic indicators for disease progression and survival. Although recent immunostimulatory methods have substantially improved analysis via conventional metaphase cytogenetics (CC), detection of chromosome changes is limited by the low mitotic activity of CLL cells in vitro. High-density single nucleotide polymorphism (SNP) arrays are commercially available technologies, which allow genome-wide detection of allelic copy number gains or losses, and loss-of-heterozygosity (LOH) regions. Aims:To assess whether SNP-arrays are more sensitive than I-FISH and CC to detect specific chromosomal abnormalities associated with prognosis in B-CLL, i.e. del13q, del11q, del17p and tri12. Methods:Blood samples from 24 patients with B-CLL at diagnosis were tested in parallel by I-FISH, CC and SNP-array. FISH: blood smear samples were hybridized with 4 probes, in order to detect deletions at 17p13.1, 11q22.3, 13q14.3, and trisomy 12 [respectively LSI p53, ATM, D13S319 and centromeric CEP12 probes, Abbott]. In normal lymphocytes, an average of 6.7% nuclei showed one signal (truncated nuclei), and we defined the cut-off level for detection of a deletion at 11% (mean+3SD). The cut-off for detection of tri12 was defined at 5% (mean+3SD). CC: blood lymphocytes are cultivated for 72 hours with immunostimulants (DSP30 and IL-2) and metaphases analyzed according to standard procedures. SNP-arrays: DNA samples (200 ng) were hybridized on the Illumina HumanCNV370-quad v3 BeadChips, which assess 373,397 markers with a median marker spacing of 4.9 kb (mean 7.8 kb). The I-Scan system was used to scan the BeadChips (primary data). GenomeStudio 1010 v1 and CNVPartition 2.4.4 package were used to process primary data and identify chromosomal deletions/amplifications and LOH regions. Results:I-FISH identified deletions at 13q (15-95% of nuclei; mean=51%), 11q (35-54%; mean=43%) and trisomy 12 (32-49%; mean=37%) in 17 (71%) [including 2 cases of biallelic deletions], 3 (12.5%) and 4 (17%) cases, respectively. No del17p was detected. Five B-CLL cases presented associated FISH abnormalities. SNP-arrays identified all changes (100%) detected by I-FISH (del13q, median size: 13 Mb – range, 0.49–50 Mb; del11q, median size: 39 Mb – range, 34.8–42 Mb]. However, SNP-arrays showed 5 additional deletions at 13q14.3. Three patients had cryptic deletions (~52 to 82 kb), not detected by the FISH LSI D13S319 probe (~130 kb) or CC, and two others had large deletions (1.1 and 33.3 Mb) but with one signal below the cut-off of 11% by I-FISH (therefore considered as negative- both had normal CC). In these two cases, tumor cell enrichment before I-FISH allowed the detection of the deletion. In one case with del13q and tri12 (30% of nuclei by FISH), an additional del11q was also detected by SNP-array and CC (in 5/24 mitoses). The size of this deletion was 37.8 Mb and involved the cytogenetic bands 11q14.1 to 11q23.2 (including ATM). In addition, SNP-arrays enabled to define more precisely the size and location of the abnormalities. For instance, in one case with tri12 (as identified by the centromeric FISH probe), a partial trisomy of chromosome 12 short arm was indeed detected by SNP-array. No 17p abnormality was detected by SNP-array (either deletion or LOH). Only one of the 24 samples had no abnormality by SNP, I-FISH or CC for the loci studied. Conclusions:Despites using a relatively low density SNP-array (~370,000 markers), a higher sensitivity of Illumina BeadChips was observed. “SNP+/FISH negative” cases can be explained by either cryptic deletions or low leukemic cell content (< cut-off level). “SNP+/CC negative” cases can be explained by either a higher resolution, or the presence of normal mitosis in excess following stimulation. Higher density SNP-arrays (> 5 million markers) may be used to improve the assay sensitivity, especially regarding del13q14 which seems to be present in a great majority of our cases. Our study confirms usefulness of SNP-arrays for prognostic evaluation of B-CLL patients at diagnosis, and suggests the use of this assay as a routine procedure. [Display omitted] Disclosures:No relevant conflicts of interest to declare.

IDH1 and IDH2 Mutations In Pediatric Acute Myeloid Leukemia

AbstractAbstract 1699Pediatric de novo acute myeloid leukemia (AML) is a heterogeneous disease that can be divided into clinically distinct subtypes based on the presence of specific chromosomal abnormalities, gene mutations, or morphologic and immunophenotypic features. The best characterized subtypes include leukemias with alterations of the gene encoding the core-binding transcription factor complex, (t(8;21)[AML1- ETO] and inv(16)/t(16;16)[CBFβ-MYH11], rearrangements of the MLL gene on chromosome 11q23, normal cytogenetics, or distinct morphology including acute promyeloctic leukemia with t(15;17)[PML-RARA] and acute megakaryoblastic leukemia (FAB-M7). In AMLs with normal cytogenetics, mutations have also been identified in a number of genes, with alterations in NPM1, FLT3 and CEBPA occurring at an appreciable frequency and influencing therapeutic responses. Recent genome-wide sequencing efforts have led to the identification of a number of new candidate genes involved in the pathogenesis of this disease. Foremost among this list are isocitrate dehydrogenase 1 (IDH1) and 2 (IDH2). IDH1 mutations were initially identified in a whole exome sequencing of glioblastoma multiforme (GBM), but were subsequently shown to be mutated in a variety of myeloid malignancies including up to 16% of adult AMLs with normal cytogenetics. The mutations in both GBM and myeloid malignancies have been heterozygous and restricted to arginine 132 in exon 4 of IDH1, or to either the homologous residue in IDH2, R172, or to a second arginine, R140, also located in its substrate binding pocket. Although the distribution of specific IDH1/IDH2 mutations varies between GBM and AML, each of the analyzed mutations result in a loss of the enzymes ability to catalyze the oxidative carboxylation of isocitrate to a-ketoglutarate (a-KG), coupled with a gain-of-function to catalyze the NADPH-dependent reduction of a–KG to 2-hydroxyglutarate (2-HG). This shift in enzymatic activity results in a dramatic increase in the levels of 2-HG within the leukemic cells; however, how the increase in this metabolite contributes to transformation remains to be determined.To investigate the frequency of IDH1 and IDH2 mutations in pediatric AML, we sequenced these genes in diagnostic samples from 227 pediatric AML patients. Our analysis identified somatic IDH1/2 mutations in 4% of cases (IDH1 N=3 and IDH2 N=5), with the frequency slightly higher in AMLs with a normal karyotype (7%). IDH1 mutations occurred in codon 132 resulting in replacement of arginine with either cysteine (N=2) or histidine (N=1). By contrast, the mutations in IDH2 did not affect the homologous residue but instead altered codon 140, resulting in replacement of an arginine with either glutamine (N=4) or tryptophan (N=1). Structural modeling studies of IDH2 suggested that the codon 140 mutations should disrupt the enzyme's ability to bind its substrate isocitrate. Consistent with this prediction, enzymology studies showed that recombinant IDH2 R140Q and R140W were unable to carry out the decarboxylation of isocitrate to α-ketoglutarate (α-KG), but instead gained the neomorphic activity to reduce α-KG to R(-)-2-hydroxyglutarate (2-HG). Analysis of primary leukemic blasts using mass spectrometry confirmed high levels of 2-HG in samples with IDH1/2 mutations. Interestingly, 3/5 leukemias also had FLT3 activating mutations, raising the possibility that these two mutations directly cooperate in leukemogenesis. Defining the biological role of the IDH1/2 mutations in leukemogenesis will benefit by a direct assessment of the biological effect of the mutations on normal murine hematopoietic cell differentiation using both in vitro and in vivo systems. Disclosures:No relevant conflicts of interest to declare.

Chromosomal abnormalities in transformed Ph‐negative myeloproliferative neoplasms are associated to the transformation subtype and independent of JAK2 and the TET2 mutations

Evolution to myelofibrosis (MF), acute myeloid leukemia or myelodysplastic syndrome (AML/MDS) may occur over time in myeloproliferative neoplasms (MPN) patients most likely due to the acquisition of additional mutations. The Groupe Francophone de cytogenetique hematologique (GFCH) has collected and reviewed 82 patients with transformation of MPN (66 AML/MDS and 16 MF). JAK2V617F and TET2 mutations were searched for in 40 and 32 patients, respectively. Significantly more -7/del(7q) (P = 0.004) and -5/del(5q) (P = 0.03) were found in AML/MDS with a higher incidence of dup1q (P = 0.01) in MF. Some specific chromosomal abnormalities occurred together, for example -5/del(5q) and -17/del(17p) (P = 0.0007). In multivariate analysis, two factors were independently associated with an inferior overall survival (OS); AML/MDS transformation (P < 0.0001) and -5/del(5q) abnormality (P = 0.02). Although both giving rise to loss of 7q, der(1;7) differed from other 7q deletions in terms of distribution (lower frequency of AML/MDS, P = 0.02), association with chromosomal abnormalities (absence of -5/del(5q), P = 0.003; increased del(20q), P = 0.05), and longer OS (P = 0.0007). We detected 24/40 (60%) JAK2V617F and 8/25 (32%) TET2 mutations in samples following transformation, ranging from wild-type to mutated forms of both genes. The mutated and wild-type forms of the genes were not found to be associated with a specific chromosomal abnormality. There was no evidence that JAK2 or TET2 mutations were associated with the type of MPN transformation, whereas the type of cytogenetic abnormalities were strongly linked, perhaps indicating that they play a specific role in the transformation process.

Cytogenetics in pre-B and B-cell acute lymphoblastic leukemia: a study of 208 patients diagnosed between 1981 and 2008

The detection of chromosome abnormalities by conventional cytogenetics, now combined with analyses using fluorescence in situ hybridization (FISH), is an important component in assessing the risk stratification of acute lymphoblastic leukemia (ALL). Identification of specific chromosome abnormalities led to the recognition of genetic subgroups based on modal chromosomal number, reciprocal translocations in B-cell ALL, or both. We report here the cytogenetic analysis of 208 patients with pre-B and B-cell ALL referred to a single laboratory between 1981 and 2008. Chromosome abnormalities were observed in 82.9% of L1/L2 ALL patients and in 83.3% of L3 patients with successful analysis at diagnosis. The proportion of diploid karyotypes tended to decrease during the period of study, from 26% to 13%, in association with technical progress and the introduction of FISH techniques. As previously reported, the incidence of high hyperdiploidy (51–67 chromosomes) was higher among children, whereas pseudodiploidy and hypodiploidy were higher among those >15 years of age. Structural chromosome abnormalities were more frequently observed among patients older than 15 years than in children (75.9% vs. 68.5%, respectively). As previously reported, t(9;22)(q34;q11) and t(12;21)(p13;q21) were the most frequent structural rearrangements among adults (26.9%) and children (19.7%), respectively. Almost 17% of the patients studied at diagnosis had further cytogenetic analyses at relapse, the majority showing clonal evolution toward a more complex karyotype. Although the detection of chromosome abnormalities by conventional cytogenetics and FISH techniques is an important tool in assessing risk stratification of ALL, some patients lack abnormalities with clinical relevance. The use of array comparative genomic hybridization (aCGH) offers an alternative for identifying copy number alterations, but cannot detect balanced chromosomal rearrangements.

Acetylation of Lysine 564 Adjacent to the C-terminal Binding Protein-binding Motif in EVI1 Is Crucial for Transcriptional Activation of GATA2

Ecotropic viral integration site 1 (EVI1) is an important transcription factor for leukemogenesis. EVI1 is a member of a group of transcription factors with C-terminal binding protein (CtBP)-binding motifs that act as transcriptional co-repressors; however, we recently found that EVI1 directly activates GATA2 transcription, which is an important gene for the maintenance of hematopoietic stem cells. We show here that EVI1-activated GATA2 transcripts derive from exon 1S of GATA2, which is specifically activated in neural and hematopoietic cells. EVI1 was acetylated by the histone acetyltransferase p300/CBP association factor (P/CAF) in myeloid leukemia cells and hematopoietic progenitor cells. Acetylation at Lys(564), which is adjacent to the CtBP-binding consensus sequence of EVI1, was found to be important for transcriptional activation of GATA2. Mutation of Lys(564) to alanine (K564A) markedly reduced the ability of EVI1 to bind DNA and activate transcription of GATA2. Furthermore, we confirmed that Lys(564) in EVI1 was specifically acetylated in leukemia and primary hematopoietic cells by using an antibody directed against an acetylated Lys(564) EVI1 peptide. Moreover, co-transfection of P/CAF with EVI1 overcame the suppressive effect of the CtBP co-repressor and resulted in GATA2 transcriptional activation; nonetheless, CtBP2 was still included in the protein complex with EVI1 and P/CAF on the EVI1-binding site in the GATA2 promoter region. Thus, acetylation of EVI1 at Lys(564) by P/CAF enhances the DNA binding capacity of EVI1 and thereby contributes to the activation of GATA2.

Oligonucleotide Array-CGH Identifies Genomic Subgroups and Prognostic Markers for Tumor Stage Mycosis Fungoides

Mycosis fungoide (MF) patients who develop tumors or extracutaneous involvement usually have a poor prognosis with no curative therapy available so far. In the present European Organization for Research and Treatment of Cancer (EORTC) multicenter study, the genomic profile of 41 skin biopsies from tumor stage MF (MFt) was analyzed using a high-resolution oligo-array comparative genomic hybridization platform. Seventy-six percent of cases showed genomic aberrations. The most common imbalances were gains of 7q33.3q35 followed by 17q21.1, 8q24.21, 9q34qter, and 10p14 and losses of 9p21.3 followed by 9q31.2, 17p13.1, 13q14.11, 6q21.3, 10p11.22, 16q23.2, and 16q24.3. Three specific chromosomal regions, 9p21.3, 8q24.21, and 10q26qter, were defined as prognostic markers showing a significant correlation with overall survival (OS) (P=0.042, 0.017, and 0.022, respectively). Moreover, we have established two MFt genomic subgroups distinguishing a stable group (0-5 DNA aberrations) and an unstable group (>5 DNA aberrations), showing that the genomic unstable group had a shorter OS (P=0.05). We therefore conclude that specific chromosomal abnormalities, such as gains of 8q24.21 (MYC) and losses of 9p21.3 (CDKN2A, CDKN2B, and MTAP) and 10q26qter (MGMT and EBF3) may have an important role in prognosis. In addition, we describe the MFt genomic instability profile, which, to our knowledge, has not been reported earlier.

Myelodysplastic Syndrome Mimicking Idiopathic Thrombocytopenic Purpura

In patients with isolated thrombocytopenia, but without significant dysplasia, diagnosis of idiopathic thrombocytopenic purpura (ITP) rather than myelodysplastic syndrome (MDS) may be taken into account. It is important to make an accurate diagnosis because different treatments are used for ITP and MDS. The purpose of this study was to investigate the clinical and hematologic features of patients who were initially diagnosed as ITP but had cytogenetic abnormalities. We retrospectively reviewed cytogenetic studies of 100 patients who were diagnosed as ITP from 2004 to 2009 at Mokdong Hospital of Ewha Womans University based on clinical features and hematologic studies. Bone marrow pathology was re-evaluated based on 2008 WHO classification. Cytogenetic analysis was performed by 24-48 hr culture of bone marrow aspirates without using mitogens and 20 metaphases were analyzed. Of the 100 patients diagnosed as ITP initially, three patients (3%) had cytogenetic abnormalities. They had no thrombocytopenia-related symptoms and thrombocytopenia was found accidentally. The numbers of megakaryocytes in bone marrow were increased and dysplasia was not found in megakaryocyte, erythroid, and myeloid cell lineages. The proportion of blasts was within normal limits. Clonal chromosomal abnormalities found were der(1;7)(q10;p10), add(9)(q12), or t(7;11)(p22;q12). Presumptive diagnosis of MDS or diagnosis of idiopathic cytopenia of undetermined significance (ICUS) was made according to 2008 WHO classification. During the follow up, disease progression was not found. In patients with suspected ITP, cytogenetic analysis should be done. If specific clonal chromosomal abnormality is found, presumptive diagnosis of MDS has to be considered and close follow up is needed.

Evidence of residual disease in cryopreserved ovarian cortex from female patients with leukemia

To systematically search for leukemic cells in cryopreserved ovarian cortex from Danish female patients with leukemia, who had ovarian cortex cryopreserved for fertility preservation before potentially sterilizing treatment. Retrospective analysis of data in a clinical project. University hospital laboratories. In total, 26 patients diagnosed with leukemia, who had ovarian tissue cryopreserved before potentially sterilizing chemotherapy and conditioning. Ovarian cortex from each patient was examined with histology and immunohistochemistry. In addition, in eight cases a specific chromosomal abnormality could be used as a genetic marker for detection of malignant cells by polymerase chain reaction (PCR). Evidence of malignant cells by immunohistochemistry or PCR. Histology and immunohistochemistry did not reveal malignant cell infiltration in the ovarian cortex of any of the patients. In six of the eight patients (75%) with chromosomal abnormalities in the malignant cells, PCR showed evidence of leukemic cells in the ovarian tissue. Immunohistochemistry was unable to locate leukemic cells in the ovarian cortex; however, PCR detected potentially malignant cells in the majority of cases. The viability and malignancy of these cells remains to be determined. At present, reimplantation of ovarian cortex to leukemia patients cannot be recommended.

Aneuploidy and DNA fragmentation in morphologically abnormal sperm

In light of the relative success of ICSI in the treatment of male infertility, much importance has been made to the selection of morphologically viable sperm. However, correlation between specific sperm morphology and chromosomal abnormalities is still relatively limited and less is known about the connection between sperm morphology and DNA integrity. Sperm obtained from isolated teratozoospermic men (n = 10) and control men (n = 9) were analysed using FISH (for chromosomes 13, 18, 21, X and Y) and TUNEL assays to determine the level of aneuploidy and DNA fragmentation. Sperm morphology was evaluated on its ability to identify the level of chromosomal abnormalities or fragmented DNA in sperm. Sperm from teratozoospermic men, compared with fertile men, had higher rates of total chromosomal abnormality (p < 0.05), total aneuploidy (p < 0.01) and chromosome 13 disomy (p < 0.01). Associations between particular types of sperm morphology and chromosomal abnormalities were observed in both control (tapered heads) and teratozoospermic (amorphous heads and tail abnormalities) samples. Levels of DNA fragmented sperm were higher in teratozoospermic men than in the control men (60.28 +/- 21.40% vs. 32.40 +/- 17.20%, p < 0.05) and positively correlated to sperm with bent necks in control samples and round heads in teratozoospermic samples (p < 0.05). Sperm of isolated teratozoospermic men have higher rates of chromosomal abnormalities and DNA fragmentation than that of the fertile controls. Specific abnormal sperm morphology can be correlated to chromosomal abnormalities and level of DNA fragmentation in sperm.

Genetic and immunophenotypic profile of IGH@ rearrangement detected by fluorescence in situ hybridization in 149 cases of B-cell chronic lymphocytic leukemia

Recent studies have shown a higher frequency of immunoglobulin heavy ( IGH@) locus rearrangement in B-cell chronic lymphocytic leukemia (B-CLL) than previously reported. However, association of the IGH@ rearrangement with specific chromosomal abnormalities and immunophenotypic markers in B-CLL is still under further investigation. In this study, we analyzed 149 bone marrow aspirate or peripheral blood specimens from patients diagnosed with B-CLL, evaluated by four different laboratory studies: morphology examination, three- or four-color flow cytometry analysis, conventional cytogenetics, and fluorescence in situ hybridization (FISH) with a dual-color, break-apart IGH@ probe in addition to a B-CLL FISH probe panel for del(11)(q22) ATM, del(13)(q14.3), del(17)(p13) TP53, and +12. An IGH@ rearrangement was found by FISH in 24 cases (16.0%). Of these 24 cases, 16 (67%) contained chromosomal abnormalities, including t(14;19)(q32;q13.2), t(8;14)(q24;q32), and t(14;18)(q32;q21). In addition, a cryptic deletion of the immunoglobulin heavy variable region ( IGHV) was revealed. Using 30% as the cutoff for positive CD38 expression, 22 of the 24 cases (92%) were positive for CD38. The present results further confirm that IGH@ rearrangement is not a rare genomic abnormality in B-CLL, and also show both that t(14;19)(q32;q13.2) is the most common cytogenetic change involving IGH@ rearrangement detected by FISH in B-CLL and that IGH@ rearrangement is correlated with CD38 expression. It is appropriate to include an IGH@ probe in the FISH panel for B-CLL diagnosis.

A New Prognostic Scoring System Including Transfusion Dependency and Cytogenetic Abnormalities for Patients with Chronic Myelomonocytic Leukemia.

Abstract 1750Poster Board I-776Chronic myelomonocytic leukemia (CMML) is a clonal disorder sharing features of both myelodysplastic syndromes (MDS) and chronic myeloproliferative disorders (MPD). The natural course of CMML is highly variable. Several small series have suggested the prognostic importance of different characteristics but a widely accepted prognostic scoring system for CMML is not available. The main aims of the study were to identify prognostic factors, including cytogenetic findings, for overall survival (OS) and acute leukemic (AL) transformation in a large series of patients with CMML and to develop an easily applicable prognostic scoring index for estimating outcome and planning treatment in the individual patient. Five hundred and seventy-two patients diagnosed of CMML according to FAB and WHO criteria in 25 centers belonging to the Spanish Registry of MDS were included in the study. Actuarial curves of OS and risk of AL evolution were built by Kaplan-Meier method and differences between curves compared with log-rank tests. Multivariate analyses of OS and risk of AL evolution were performed by Cox proportional hazards regression method. The weights assigned to the variables included in the final prognostic scoring system were based on the regression coefficients from the proportional hazards models. Median age was 73 yr and 397 (69%) were males. According to FAB criteria 61% of the patients had MDS-CMML (absolute WBC count '13 × 109/L) and 39% MPD-CMML and by WHO classification 86% were CMML-1 (blasts <10% in bone marrow and <5% in blood) and 14% CMML-2. Karyotype was available in 419 patients (abnormal in 113) and RBC transfusion dependency in 415 (222 transfusion-dependent). With a median follow-up of 33 months, the median OS was 25 months. Seventy-five patients evolved to AL and the cumulative risk to AL transformation was 25% at 5 yr. In univariate analyses patients with CMML-2, presence of 2–3 cytopenias, MPD-CMML, hemoglobin <10 g/L, poor-risk cytogenetics (defined as trisomy 8 or complex karyotype), and RBC transfusion dependency had both a shorter OS and higher risk of AL evolution (P<0.001 in all cases). Additionally, patients with serum LDH level >480 U/L (P<0.001), monocytes >3 × 109/L (P<0.001), platelets '100 × 109/L (P=0.001), serum ferritin level >500 ng/mL (P<0.01), and males (P=0.01) also had a poorer OS. In multivariate analyses the most relevant variables for OS were WHO (P<0.001) and FAB classifications (P<0.001), RBC transfusion dependency (P<0.001), LDH level (P<0.001), hemoglobin level (P<0.001), and cytogenetics (P=0.001). For AL transformation risk the only independent factors were WHO (P<0.001) and FAB classifications (P<0.001), RBC transfusion dependency (P<0.001), and cytogenetics (P=0.01). A prognostic scoring system using WHO (CMML-2, 1.5 points) and FAB classifications (MPD-CMML, 1 point), RBC transfusion dependency (dependent, 1 point), LDH level (>480 U/L, 1 point) and cytogenetic risk group (high-risk, 1 point) was developed (overall risk, 0 – 5.5 points). This scoring system was able to stratify patients into 4 risk groups (low, 0 points; intermediate-1, 1–1.5 points; intermediate-2, 2 points; and high, 2.5 – 5.5 points) with significantly different probabilities of death (median OS, 105, 38, 22, and 11 mo, respectively; P<0.001; Figure) and AL evolution (cumulative risk at 5 yr, 3%, 24%, 33%, and 40%, respectively; P=0.001). The results of this study confirm the prognostic impact of FAB and WHO subtypes and serum LDH level and recognize for the first time the relevance of RBC transfusion dependency and specific chromosomal abnormalities in CMML. Finally, they offer a simple and powerful index for assessing prognosis and planning therapy in CMML. [Display omitted] DisclosuresNo relevant conflicts of interest to declare.