Exome Sequencing Reveals The Genetic Landscape Of Rare Atypical e6a2 CML

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 3995-3995
Author(s):  
Arati V. Rao ◽  
Yuri D. Fedoriw ◽  
Kristy L. Richards ◽  
Zhen Sun ◽  
Cassandra L Love ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
High Throughput Sequencing ◽  
Conflicts Of Interest ◽  
Gene Mutations ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Cancer Genes ◽  
Sequencing Data ◽  
Illumina Platform ◽  
Depth Analysis

Abstract Background Over 90% of Ph-positive chronic myelogenous leukemia (“typical CML”) patients have breakpoints in the M-bcr, which typically result in b2a2 (e13a2) and/or b3a2 (e14a2) fusion mRNAs, both of which are translated into the p210 BCR-ABL protein. CML patients with the p190 BCR-ABL (m-bcr) or p230 BCR-ABL (μ-bcr) fusion genes have been reported. Atypical BCR breakpoints outside these cluster regions are extremely rare. For instance, only 8 cases have been described of e6a2 fusion CML. Very little is known about the clinical or biological characteristics of this subtype of CML, including the role of collaborating gene mutations in the development of disease. In this study, we defined the gene mutations that occurred in a rare e6a2 CML case and compared the observed gene mutations to those in “typical” chronic phase (CP)-CML cases. To our knowledge, this is the first comparison of the genetic mutations occurring in typical CML and in this rare atypical form of CML. Methodology We identified the index e6a2 CML patient, and eight additional typical CML patients for whom we had bone marrow aspirate, peripheral blood and paired normal tissue. We performed whole-exome sequencing for all of these samples using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 3 GB of sequencing data using high throughput sequencing on the Illumina platform. Results We identified 15 candidate cancer genes that were somatically mutated in our e6a2 CML patient. Commonly implicated biological processes comprising these genes included transcription (STAT5A, TET2, GTF2F1), cellular differentiation (TP73), and signal transduction (GPR116). Interestingly, the majority of these mutations also occurred in typical CML, albeit at lower frequency. Thus, genes mutated common to our atypical case and typical CMLs included STAT5A, TET2, GTF2F1, ABL1 and CYP2A6. Thus, while atypical e6a2 BCR-ABL fusion CML cases are extremely rare, they appear to share many aspects of the biology with typical CMLs. Conclusion This study represents an in-depth analysis of a rare e6a2 CML in combination with one of the first analyses of gene mutations that occur in typical CML. Our data provide a significant first step to identifying genes that play a role in the pathogenesis along with BCR-ABL that perhaps contribute to drug resistance, and ultimately impact overall survival. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Mutations of the N-ras gene in juvenile chronic myelogenous leukemia

Blood ◽  
1994 ◽  
Vol 83 (8) ◽  
pp. 2248-2254 ◽  
Author(s):  
J Miyauchi ◽  
M Asada ◽  
M Sasaki ◽  
Y Tsunematsu ◽  
S Kojima ◽  
...  
Keyword(s):  
Sequence Analysis ◽  
Chronic Myelogenous Leukemia ◽  
Blast Crisis ◽  
Gene Mutations ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Disease Stage ◽  
Nucleotide Sequence Analysis ◽  
Age Group ◽  
Ras Gene

Juvenile chronic myelogenous leukemia (JCML), a myeloproliferative disorder of childhood, is distinct from adult-type chronic myelogenous leukemia (CML) and bears resemblance to chronic myelomonocytic leukemia (CMMoL). Since mutations in the N-ras gene have been found at high frequencies in CMMoL, but only rarely in CML, we analyzed mutations activating the N-ras gene in 20 patients with JCML. We used the strategy for analysis of gene mutations based on in vitro DNA amplification by polymerase chain reaction (PCR) followed by single- strand conformation polymorphism (SSCP) analysis and/or direct sequence analysis. Nucleotide sequence analysis showed single nucleotide substitutions involving codons 12, 13, or 61 in six of 20 patients (30%). Four of six patients with mutations were in chronic phase and the other two in blast crisis, indicating no apparent correlation with disease stage. Most of the patients with mutations were in the older age group with poor prognosis, although one patient in the younger age group also harbored the mutation. These data suggest that N-ras gene mutations may be involved in the pathogenesis and/or prognosis of JCML and provide further evidence that JCML is an entity distinct from CML.

scholarly journals Challenges in identifying cancer genes by analysis of exome sequencing data

2016 ◽  
Vol 7 (1) ◽  
Author(s):  
Matan Hofree ◽  
Hannah Carter ◽  
Jason F. Kreisberg ◽  
Sourav Bandyopadhyay ◽  
Paul S. Mischel ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Cancer Genes ◽  
Sequencing Data ◽  
Exome Sequencing Data

Whole Exome Sequencing Reveals Spectrum of Gene Mutations in Pediatric AML

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 124-124
Author(s):  
Norio Shiba ◽  
Kenichi Yoshida ◽  
Yusuke Okuno ◽  
Yuichi Shiraishi ◽  
Yasunobu Nagata ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
Whole Exome Sequencing ◽  
Conflicts Of Interest ◽  
Massively Parallel Sequencing ◽  
Gene Mutations ◽  
Pediatric Leukemia ◽  
Protein Coding ◽  
Whole Exome ◽  
Recurrent Mutations ◽  
Pediatric Aml

Abstract Abstract 124 Background Pediatric acute myeloid leukemia (AML) comprises ∼20% of pediatric leukemia, representing one of the major therapeutic challenges in pediatric oncology with the current overall survival remains to be ∼60%. As for the molecular pathogenesis of pediatric AML, it has been well established that gene fusions generated by recurrent chromosomal translocations, including t(15;17), t(8;21), inv(16) and t(9;11), play critical roles in leukemogenesis. However, they are not sufficient for leukemogenesis, indicating apparent need of additional genetic hits, and approximately 20% of pediatric AML cases lack any detectable chromosomal abnormalities (normal karyotype AML). Currently, a number of gene mutations have been implicated in the pathogenesis of both adult and pediatric AML, including mutations of RAS, KIT and FLT3, and more recently, a new class of mutational targets have been reported in adult AML, including CEBPA, NPM1, DNMT3A, IDH1/2, TET2 and EZH2. However, mutations of the latter class of gene targets seem to be rare in pediatric AML cases, whereas other abnormalities such as a NUP98-NSD1 fusion are barely found in adult cases, indicating the discrete pathogenesis between both AML at least in their subsets. Meanwhile, the recent development of massively parallel sequencing technologies has provided a new opportunity to discover genetic changes across the entire genomes or protein-coding sequences in human cancers at a single-nucleotide level, which could be successfully applied to the genetic analysis of pediatric AML to obtain a better understanding of its pathogenesis. Methods In order to reveal a complete registry of gene mutations and other genetic lesions, we performed whole exome sequencing of paired tumor-normal specimens from 23 pediatric AML cases using Illumina HiSeq 2000. Although incapable of detecting non-coding mutations and gene rearrangements, the whole-exome approach is a well-established strategy for obtaining comprehensive spectrum of protein-coding mutations. Recurrently mutated genes were further examined for mutations in an extended cohort of 200 pediatric AML samples, using deep sequencing, in which the prevalence and relative allele frequencies of mutations were investigated. Results Whole-exome sequencing of paired tumor-normal DNA from 23 patients were analyzed with a mean coverage of more than x120, and 90 % of the target sequences were analyzed at more than x20 depth on average. A total of 237 somatic mutations or 10.3 mutations per sample were identified. Many of the recurrent mutations identified in this study involved previously reported targets in adult AML, such as FLT3, CEBPA, KIT, CBL, NRAS, WT1, MLL3, BCOR, BCORL1, EZH2, and major cohesin components including XXX and ZZZ. On the other hand, several genes were newly identified in the current study, including BRAF, CUL2 and COL4A5, which were validated for the clinical significance in an extended cohort of 200 pediatric cases. Discussion Whole exome sequencing unmasked a complexity of gene mutations in pediatric AML genomes. Our results indicated that a subset of pediatric AML represents a discrete entity that could be discriminated from the adult counterpart, in terms of the spectrum of gene mutations. Disclosures: No relevant conflicts of interest to declare.

Whole Genome and Exome Sequencing Reveals the Genetic Landscape of Burkitt Lymphoma

Blood ◽  
2011 ◽  
Vol 118 (21) ◽  
pp. 433-433
Author(s):  
Cassandra L Love ◽  
Dereje Jima ◽  
Jenny Zhang ◽  
Vladimir Grubor ◽  
Rodney R. Miles ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
High Throughput ◽  
Burkitt Lymphoma ◽  
Normal Tissue ◽  
High Throughput Sequencing ◽  
B Cell Lymphoma ◽  
Chromosomal Translocations ◽  
Cancer Genes ◽  
Genetic Causes ◽  
Paired Normal Tissue

Abstract Abstract 433 Burkitt lymphoma (BL) is a relatively uncommon lymphoma, but is clinically important because it is curable when diagnosed properly. BL is also an important model disease for studying cancer. Chromosomal translocations of the MYC gene are a defining feature of BL. Diffuse large B cell lymphoma (DLBCL) is the most common form of lymphoma in adults and demonstrates overlapping morphology, immunophenotype and clinical behavior with BL. The genetic causes and the role of specific mutations in BL are largely unknown. Th e decoding of the human genome and the advent of high-throughput sequencing have provided rich opportunities for the comprehensive identification of the genetic causes of cancer. We began by sequencing 2 complete lymphoma tumor genomes (and paired normal tissue) derived from DLBCL and BL respectively. The pattern of somatic base alterations in both DLBCL and BL genomes indicated a predominance of G→A/C→T and A→G/T→C transitions (P<10−6) suggesting that the majority of these mutations arise from endogenous processes rather than environmental exposures, as has been observed with lung cancer and tobacco. In order to comprehensively identify genes that are recurrently mutated in DLBCL and BL, we obtained a total of 95 cases of DLBCLs and 60 cases of BL. The DLBCL cases were divided into a discovery set (N=34) and a prevalence set (N=61). The Burkitt cases were also divided into discovery and prevalence sets (N=15, N=45 respectively). For each of the discovery set cases we also obtained paired normal tissue. We performed whole-exome sequencing for all of these using the Agilent solution-based system of exon capture, which uses RNA baits to target all protein coding genes (CCDS database), as well as ∼700 human miRNAs from miRBase (v13). In all, we generated over 6 GB of sequencing data using high throughput sequencing on the Illumina platform. We identified 525 candidate cancer genes that were recurrently somatically mutated in DLBCL and BL. We found that each tumor had an average of 20 gene alterations, which is fewer than most other solid tumors sequenced to date. Commonly implicated biological processes comprising these genes included signal transduction (e.g. PIK3CD, PDGFRA) and chromatin modification (e.g. MLL3, SETD2), affecting 17.2% and 14.8% of the total genetic events respectively. We found several genes related to cancer that were commonly mutated in both BL and DLBCL, including MYC, BTG1 and SETD2. Mutations in MYC were much more common in BL compared to DLBCL, suggesting that mutation of MYC might serve as an independent oncogenic mechanism in BL, in addition to chromosomal translocations. Many known cancer genes were found to be exclusively mutated in BL including SMARCA4, a gene known to regulate the expression of CD44 which is implicated in tumorigenesis. This study represents one of the first in-depth analyses of a BL genome and one of the largest applications of exome sequencing in cancer. Our data provide the most comprehensive genetic portrait of human BL to date, and provides a significant first step to identifying the genetic causes of the disease. Disclosures: No relevant conflicts of interest to declare.

Clonal Evolution and Devolution Following Chemotherapy in Adult Acute Myelogenous Leukemia.

Blood ◽  
2012 ◽  
Vol 120 (21) ◽  
pp. 2487-2487
Author(s):  
Brian Parkin ◽  
Peter Ouillette ◽  
Yifeng Li ◽  
Cheng Li ◽  
Kerby Shedden ◽  
...  
Keyword(s):  
Complete Remission ◽  
Gene Mutation ◽  
Induction Chemotherapy ◽  
Acute Myelogenous Leukemia ◽  
Conflicts Of Interest ◽  
Gene Mutations ◽  
Myelogenous Leukemia ◽  
Stable Gene ◽  
Paired Data ◽  
Acute Myelogenous

Abstract Abstract 2487 Introduction: Despite significant advances in the understanding of the biology of adult acute myelogenous leukemia (AML), overall survival remains poor due chiefly to the high rate of relapse after achieving complete remission as well as primary failure of induction chemotherapy. Efforts to further unravel the mechanisms leading to relapse and primary refractory disease are critical in order to guide the development of effective and durable treatment strategies for AML. To that end, this study seeks to elucidate the clonal relationship of AML in various disease phases. Methods: We employed SNP 6.0 array-based genomic profiling of acquired copy number aberrations (aCNA) and copy neutral LOH (cnLOH) together with sequence analysis of recurrently mutated genes to characterize paired AML genomes. We analyzed 28 AML sample pairs from patients that achieved complete remission with chemotherapy and subsequently relapsed (median remission duration 272 days [range 25 – 1249 days]) and 11 sample pairs from patients with persistent disease following induction chemotherapy. AML cell samples were isolated with a Ficoll gradient, negatively selected using Miltenyi microbead columns, and then further purified with flow cytometric cell sorting. Processed DNA isolated from highly purified AML blasts and paired buccal DNA was hybridized to Affymetrix SNP 6.0 arrays. aCNA were visually identified using the dChip program in paired data displays and corroborated by algorithmic lesion scoring, and cnLOH was detected using internally developed software. In addition, 11 genes known to be recurrently mutated in AML (CEBPA, DNMT3A, IDH1, IDH2, RUNX1, BCORL1, NPM1, NRAS, KRAS, FLT3 and TP53) were resequenced in all 39 presentation samples to identify somatically acquired mutations. Genes found mutated in individual AML cases were subsequently tested for the persistence of the mutation in paired samples. Results: For the 28 paired specimens in the relapsed cohort, comparison of aCNA and cnLOH occurrences, gene mutation patterns and karyotypes revealed 6 cases that carried no aCNA/cnLOH at either presentation or relapse, but at presentation carried at least 1 gene mutation, all of which but one were stable in relapse (1 case lost a RUNX1 mutation but carried a t(8;21) in both disease stages); 11 cases that were characterized by the presence of aCNA/cnLOH at presentation, of which 55% (6 of 11) gained additional aCNA/cnLOH at relapse; 6 cases without aCNA/cnLOH at presentation that gained aCNA/cnLOH at relapse, of which 2 concurrently lost a FLT3-ITD or CEPBA mutation; and 5 cases that carried no informative genomic events. For the 11 paired specimens in the persistent AML cohort, the same comparison revealed 2 cases without aCNA/cnLOH before or after chemotherapy and stable gene mutations; 5 cases with aCNA/cnLOH at presentation that carried the same genomic lesions and gene mutations before and after chemotherapy; 3 cases with aCNA/cnLOH present at enrollment that lost some but not all of these aCNA/cnLOH and gained none after initial induction therapy; and 1 additional case that lost a FLT3-ITD. Comparative analysis of these patterns demonstrates that relapsed AML invariably represents reemergence or evolution of an antecedent clone. Furthermore, all individual aCNA or cnLOH detected at presentation persisted at relapse indicating that this lesion type is proximally involved in AML evolution. Analysis of informative paired persistent AML disease samples uncovered at least two coexisting dominant clones of which at least one was chemotherapy sensitive and one resistant. Conclusion: This detailed genomic analysis supports the conclusion that incomplete eradication of AML founder clones rather than stochastic emergence of fully unrelated novel clones underlies AML relapse and persistence with direct implications for clinical AML research. Disclosures: No relevant conflicts of interest to declare.

GADD45a Is a Tumor Suppressor In BCR-ABL-Driven Leukemogenesis

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 1467-1467
Author(s):  
Kaushiki Mukherjee ◽  
Xiaojin Sha ◽  
Ravi Bhatia ◽  
Tomasz Skorski ◽  
Barbara Hoffman ◽  
...  
Keyword(s):  
Tumor Suppressor ◽  
Kinase Inhibitor ◽  
Conflicts Of Interest ◽  
Multiple Stressors ◽  
Blast Crisis ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Pcr Analysis ◽  
M Cell ◽  
Number Of Patients

Abstract The BCR-ABL fusion oncogene which encodes a fused deregulated tyrosine kinase causes chronic myelogenous leukemia (CML) in humans. Imatinib, a small molecule ABL kinase inhibitor has been highly effective in treating chronic phase (CP) CML patients. However, a substantial number of patients undergo relapse due to development of resistance to imatinib therapy that leads to blast crisis (BC-CML), which is invariably fatal within weeks to months. Additional genetic aberrations assist in progression and identification of key players that are responsible for transformation is of utmost importance from a therapeutic point of view. Growth arrest DNA damage 45a (Gadd45a) gene, a member in the gadd45 family of genes including Gadd45b & Gadd45g, was identified as a myeloid differentiation primary response gene. There is evidence consistent with it’s involvement in G2/M cell cycle arrest and apoptosis in response to multiple stressors, including genotoxic and oncogenic stress. To investigate the effect of Gadd45a in the development of CML, adaptive bone marrow transplantation experiments with either wild type or Gadd45a null myeloid progenitors expressing 210-kD BCR-ABL fusion oncoprotein revealed that loss of Gadd45a accelerated BCR-ABL driven CML resulting in the development of a more aggressive AML/BC like disease. Recent newly obtained data indicate that number of Gadd45a deficient Leukemic stem cells (LSC) harboring BCR-ABL increased as disease progressed confirming Gadd45a as a crucial tumor suppressor in CML. Recent data also indicate, that transformed Gadd45a deficient progenitors exhibit increased proliferation and decreased apoptosis, associated with enhanced PI3K-AKT-mTOR-4E-BP1 signaling and upregulated oncogenic p30C/EBPα. More importantly, newly obtained data indicate that Gadd45a transcript levels in peripheral blood of human blast crisis (BC-CML) samples was found to be reduced compared to accelerated phase (AP-CML), chronic phase (CP-CML) and normal controls, assessed by Quantitative real time PCR analysis. Collectivly these data strongly suggest that Gadd45a expression is a novel prognostic indicator of CML progression, implicating Gadd45a as a downregulated target of BCR-ABL associated with progression to more aggressive stages. To conclude, our findings provide novel evidence that Gadd45a functions as a suppressor of BCR/ABL driven myeloid leukemogenesis, & that suppresion of Gadd45a is associated with CML progression. These data provide the impetus to further elucidate the role Gadd45a plays in suppressing the development of CML, and explore how its loss contributes to the progression of CML to more aggressive leukemic phenotypes. Disclosures: No relevant conflicts of interest to declare.

Comparison Of Mutational Profiles Of Diagnosis and Relapsed Pediatric B-Acute Lymphoblastic Leukemia: A Report From The COG ALL Target Project

Blood ◽  
2013 ◽  
Vol 122 (21) ◽  
pp. 824-824 ◽  
Author(s):  
Jinghui Zhang ◽  
Mignon L. Loh ◽  
Xiaotu Ma ◽  
Michael Rusch ◽  
Gang Wu ◽  
...  
Keyword(s):  
Acute Lymphoblastic Leukemia ◽  
Exome Sequencing ◽  
Somatic Mutations ◽  
Conflicts Of Interest ◽  
Lymphoblastic Leukemia ◽  
Therapeutic Targets ◽  
Purine Metabolism ◽  
White Blood Count ◽  
Ras Signaling ◽  
Sequencing Data

Abstract Characterization of the genetic landscape of relapsed pediatric acute lymphoblastic leukemia (ALL) and changes that occur with disease progression provides insight into the molecular basis of relapse and may identify new therapeutic targets. We analyzed 20 diagnosis-remission (germline)-relapse trio samples of pediatric B-ALL by high-coverage (>200x) whole-exome sequencing. Included patients were originally NCI high risk (HR) by either age (≥10 years) or white blood count (≥50,000/microliter), enrolled on a Children’s Oncology Group B-ALL trial, and experienced bone marrow relapse. Samples were selected based upon availability of sufficient high quality material from all three time points. We identified recurrent relapse-specific somatic mutations in 5 genes with significant frequency found in genes encoding the purine 5’ nucleotidase NT5C2 (n=7, 35%) and the histone acetyltransferase CREBBP (n=2, 10%). Furthermore, we discovered novel recurrent somatic mutations that were highly enriched in relapsed ALL (20%) compared with diagnosis (5%) in WHSC1, USH2A and NT5C1B, another enzyme involved in purine metabolism. Three of the four WHSC1 mutations cause the same amino acid change E1099K in the highly conserved SET domain in which structural modeling predicts perturbed WHSC1-substrate interactions resulting in increased WHSC1 activity. The WHSC1 and NT5C2 mutations are mutually exclusive with a combined prevalence of 55% in relapsed tumors. Analysis of a validation cohort of 63 independent trios from both NCI standard risk and HR cohorts replicated the findings in NT5C2 (n=8, 13%) and WHSC1 E1099K (n=6, 10%). Five pathways were significantly mutated at relapse with high-frequency somatic mutations present at diagnosis and/or relapse of the 20 ALL trio samples: the Ras signaling pathway (NRAS, KRAS, PTPN11, FLT3; 65%), genes involved in histone modification (MLL2, WHSC1, SETD2, CREBBP; 50%), purine metabolism (NT5C2 and NT5C1B; 45%), tyrosine kinase signaling (JAK2, CRLF2; 25%) and genes regulating B-cell development (PAX5, IKZF1; 15%). The median number of non-silent coding region sequence mutations in diagnostic samples was 10 (range 4-25) while that of the relapse samples was 25 (range 7-506) including 3 hypermutated samples with >100 non-silent mutations accompanied by a dominance of C(G)>T(A) substitution in relapse-specific mutations, suggesting a possibility that these mutations may be induced by a specific mutagen. Most of the diagnostic and relapse tumors were polyclonal based on diagnosis-relapse comparison of mutant allele fraction (MAF). Inter-tumor MAF of a recurrently mutated gene was highly heterogeneous despite an estimated >70% leukemia involvement for most specimens, suggesting presence of subclonal mutations. For example, NT5C2 MAF in the relapsed specimens ranges from a low of 0.08 to a high of 0.93. Ten relapsed specimens demonstrated evolution from a minor subclone (<10%) in the diagnostic specimen; 8 of the subclones have oncogenic mutations in NRAS, KRAS, JAK2, WHSC1 or CRLF2. A notable finding was the lack of preservation of specific clonal RAS pathway mutations from diagnosis to relapse as subclonal mutations in KRAS, PTPN11 and FLT3 present in diagnosis were replaced by a dominant NRAS mutation in relapse. We were also able to identify structural alterations present in both relapse and diagnosis tumors from exome sequencing data, including IGH@-CRLF2 fusion (n=2), BTG1 deletion (n=2), ETV6-RUNX1 fusion (n=1), intragenic deletion of RUNX1 (n=1) and MAP3K2 (n=1), focal amplification in the last exon of MYC (n=1) and a t(1;14)(p36;q32) translocation resulting in truncation of SLC2A5 and BTBD7. These results provide new insights into the genetic events contributing to the relapse of pediatric B-ALL, and suggest new potential therapeutic targets. Disclosures: No relevant conflicts of interest to declare.

scholarly journals Modeling Read Counts for CNV Detection in Exome Sequencing Data

2011 ◽  
Vol 10 (1) ◽  
Author(s):  
Michael I. Love ◽  
Alena Myšičková ◽  
Ruping Sun ◽  
Vera Kalscheuer ◽  
Martin Vingron ◽  
...  
Keyword(s):  
Exome Sequencing ◽  
High Throughput Sequencing ◽  
Gc Content ◽  
High Sensitivity ◽  
Read Depth ◽  
Large Chromosome ◽  
Sequencing Data ◽  
Sequencing Project ◽  
Exome Sequencing Data ◽  
Control Set

Varying depth of high-throughput sequencing reads along a chromosome makes it possible to observe copy number variants (CNVs) in a sample relative to a reference. In exome and other targeted sequencing projects, technical factors increase variation in read depth while reducing the number of observed locations, adding difficulty to the problem of identifying CNVs. We present a hidden Markov model for detecting CNVs from raw read count data, using background read depth from a control set as well as other positional covariates such as GC-content. The model, exomeCopy, is applied to a large chromosome X exome sequencing project identifying a list of large unique CNVs. CNVs predicted by the model and experimentally validated are then recovered using a cross-platform control set from publicly available exome sequencing data. Simulations show high sensitivity for detecting heterozygous and homozygous CNVs, outperforming normalization and state-of-the-art segmentation methods.

Clinical Usefulness and Safety of Peripherally-Inserted Central Catethers (PICC) in Hematological Patients.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 4545-4545
Author(s):  
Giacomo Salvatore Morano ◽  
Caterina Mercanti ◽  
Vincenzo Federico ◽  
Angela Matturro ◽  
Alessandra Micozzi ◽  
...  
Keyword(s):  
Hodgkin Lymphoma ◽  
Conflicts Of Interest ◽  
Wide Spectrum ◽  
Lymphoblastic Leukemia ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Haematological Malignancies ◽  
Promising Alternative ◽  
Advanced Phase ◽  
Median Period

Abstract Abstract 4545 The use of Peripherally-Inserted Central Catheters (PICC) as an alternative to Central Venous Catethers (CVC) is becoming very frequent in different setting of patients. To highlight the role of PICC also in patients with haematological malignancies, we revised our single Institute experience from 11/2008 to 7/2009. On the whole, 33 PICCs (BARD Groshong 4 Fr) were inserted in 32 patients [M/F 11/21, median age 59.9 years, Interquartile Range (IR) 47.1 – 74.7] for a total number of 1979 days. Twelve patients had Acute Myelogenous Leukemia (AML), 3 Acute Lymphoblastic Leukemia (ALL), 6 Non-Hodgkin Lymphoma (NHL), 5 Hodgkin Lymphoma (HD), 6 Myelodysplastic/Myeloproliferative Disorders (MDS/MPD); as to disease phase, 6 patients were at onset, 10 in complete response (3 before consolidation therapy and 7 before autologous peripheral stem-cell transplantation), 5 at disease relapse, 7 in chronic phase with transfusional requirement and 4 in advanced phase. PICC was successfully inserted in all cases with US-guide (in 21 cases via basilica vein, in 11 via brachial vein and in 1 via cephalic vein). At insertion, platelets count was < 50 × 109/l in 17/33 cases (51.5%) while WBC count was < 1.0 × 109/l in 6/33 cases (18.1%). An accidental PICC extraction occurred after 13 days; in addition, there were 2/33 (6.0%) (0,03/1000 gg) thrombophlebitic complications after 15 and 21 days respectively and 6/33 (18.1%)(0.10/1000 gg) infective complications [4 sepsis catheter-related from Staphylococci (3) or Acromobacter (1) and 2 local flogistic infiltration]. On the whole, 17/33 PICCs (51.5%) were removed after a median period of 39 days (IR 17 – 64); the reasons for removal were completion of treatment in 4 patients, death unrelated to the PICC in 6 and catheter-related complications in 7 (5 for infection, 1 for thrombosis and 1 for accidental extraction). The remaining 16/33 PICCs (48.5%) are still in use after a median period of 73 days (IR 30 – 93). In conclusion, PICC seems to be a useful, safe and promising alternative to conventional CVC for many haematological malignancies in a wide spectrum of clinical settings, ranging from intensive chemotherapy (including autotransplant procedure) to chronic management and very advanced phases requiring palliative approach. Disclosures: No relevant conflicts of interest to declare.

The BCR-ABL1-Regulated hnRNP A1, hnRNP E2, and hnRNP K Are Differentially Expressed Between CD34+ and CD34+/CD38- Ph+ Cells, and After Blastic Transformation of CML.

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 1222-1222
Author(s):  
Jason G Harb ◽  
Paolo Neviani ◽  
Joshua J Oaks ◽  
Guido Marcucci ◽  
Peter Hokland ◽  
...  
Keyword(s):  
Stem Cells ◽  
Disease Progression ◽  
Cell Line ◽  
Rna Binding ◽  
Conflicts Of Interest ◽  
Rna Binding Proteins ◽  
Chronic Phase ◽  
Myelogenous Leukemia ◽  
Hnrnp A1 ◽  
Hnrnp K

Abstract Abstract 1222 The molecular mechanism leading to disease progression of chronic myelogenous leukemia (CML) still remains to be identified although enhanced BCR/ABL expression and activity seems to play an important role in controlling genomic stability, differentiation, and self renewal of the leukemic cell clone undergoing blastic tranformation by affecting expression and function of RNA binding proteins (RBPs) like hnRNP A1, hnRNP E2, and hnRNP K (Perrotti D. et al. J. Clin Invest. 2010). We previously reported (Harb J. et al., ASH 2009) that a BCR-ABL1 dosage-dependence and hierarchical organization exists for the expression of hnRNP A1, hnRNP E2, and hnRNP K in cell line models of CML. In fact, as BCR/ABL levels increase, upregulated expression of hnRNP A1 is observed, followed by increased expression of hnRNP E2 and hnRNP K. HnRNP A1 and hnRNP K were also temporally expressed within Lineage- Sca-1+ c-kit+ (LSK), common myeloid progenitors (CMP), and granulocyte macrophage progenitors (GMP) in a mouse model (SCLtTA TRE-BCR/ABL) of chronic myeloid leukemia. Interestingly, hnRNP A1 and hnRNP K levels in BCR/ABL+ mouse progenitors correlated with disease severity as mice with higher levels of these RBPs presented a more progressed phenotype characterized by increased mixed lineage B220+/Mac-1+ progenitors in bone marrow and spleen when compared with mice that developed a CML-CP-like phenotype. Here we show that hnRNP A1, hnRNP E2, and hnRNP K expression levels, as well as BCR/ABL activity are different in HSC (CD34+/CD38-), CMP (CD34+/CD38+/CD45+/IL-3Ra-), and GMP (CD34+/CD38+/CD45+/IL-3Ra+) isolated from peripheral blood of patients in chronic phase (CML-CP) at diagnosis, untreated accelerated phase (CML-AP), and blast crisis (CML-BC). Interestingly, in CML-CP, the highest expression of hnRNP A1 was found in the CD34+/CD38- stem cell fraction and it gradually decreased in the more mature CMP and GMP progenitors (55% and 65% lower, respectively). By contrast, consistent with the role of hnRNP A1 as regulator of progenitor cell proliferation and survival, hnRNP A1 expression progressively increased in the HSC, CMP and GMP fractions isolated from patients in CML-AP and CML-BC. Unlike hnRNP A1, hnRNP E2 and hnRNP K were barely detected in the CD34+/CD38- from CML-CP patients but their expression was markedly pronounced in the HSC fraction of progressed CML patients. In agreement with our cell line data, expression of hnRNP A1 and hnRNP E2 in advanced CML (CML-AP and CML-BC) increases when CD34+/CD38- stem cells undergo maturation toward CMP. Conversely, it appears that hnRNP K expression is the last to increase in CML-BC, suggesting a hierarchical regulation of RBP expression during differentiation and lineage commitment. Expectedly, levels of hnRNP A1 in CMPs increase during disease progression (CP<AP<BC). Because of the highest expression of hnRNP A1 in the CD34+/CD38- fraction in CML-CP, and its previously described role in regulating survival and proliferation of Ph+ CD34+ cells (Iervolino et al., Mol Cell Biol 2002; Neviani et al. Cancer Cell 2005), we lentivirally transduced a hnRNP A1 shRNA into the CD34+/CD38- and CD34+/CD38+ cell fractions in CML-CP and assessed the role played by this RBP in survival of primitive and committed CML-CP progenitors. Western blot analysis demonstrated that hnRNP A1 was efficiently knockdown (≥ 80% reduction) in both compartments. Due to its ability to regulate BCR-ABL1 expression/function through the SET-PP2A axis, downregulation of hnRNP A1 led to reduced BCR/ABL activity in both stem and progenitor cells. However, cell survival and cytokine-dependent proliferation were severely compromised in committed progenitors transduced with the hnRNP A1 shRNA but not in the shRNA hnRNP A1-expressing CD34+/CD38- cells, consistent with the notion that progenitors but not stem cells are BCR-ABL1 oncogene addicted. Colony forming assays performed with empty vector- and hnRNP A1 shRNA-transduced CD34+/CD38- cells showed a 50% reduction in the number of CFCs upon hnRNP A1 downregulation. Accordingly, levels of the hnRNP A1-regulated anti-apoptotic Bcl-xL were substantially reduced in CD34+/CD38- cells. Taken together, these data further implicate RBPs hnRNP A1, hnRNP E2, and hnRNP K in CML disease progression and suggest their possible role in the control of survival of stem and primitive CML-CP progenitors. Disclosures: No relevant conflicts of interest to declare.

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