13. Clinical utility of mate pair sequencing to detect diagnostic and prognostic chromosomal rearrangements and copy number changes in patients with acute myeloid leukemia

2018 ◽  
Vol 224-225 ◽  
pp. 55
Author(s):  
Linda B. Baughn ◽  
Stephanie A. Smoley ◽  
Umut Aypar ◽  
Beth A. Pitel ◽  
George Vasmatzis ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Clinical Utility ◽  
Chromosomal Rearrangements ◽  
Mate Pair ◽  
Copy Number Changes ◽  
Acute Myeloid

scholarly journals Simultaneous FLT3, NPM1 and DNMT3A mutations in adult patients with acute myeloid leukemia – case study

2019 ◽  
Vol 27 (3) ◽  
pp. 245-254 ◽  
Author(s):  
Florin Tripon ◽  
George Andrei Crauciuc ◽  
Valeriu George Moldovan ◽  
Alina Bogliș ◽  
István Benedek ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cytogenetic Analysis ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Somatic Mutations ◽  
Molecular Technique ◽  
Chromosomal Anomalies ◽  
Copy Number Changes ◽  
Next Generation Sequencing Ngs ◽  
Acute Myeloid

Abstract Background: Nowadays, cytogenetics and molecular genetics, but not only, are mandatory in acute myeloid leukemia (AML) management, as a consequence of their impact on AML pathogenesis, classification, risk-stratification, prognosis and treatment. Objective: The aim of our study was to present our algorithm for the analysis of copy number changes, aneuploidies and somatic mutations focusing on a rare AML case positive for four somatic mutations. Methods: Cytogenetic analysis, Multiplex Ligationdependent Probe Amplification (MLPA) analysis, somatic mutation analysis (for FLT3 ITD, FLT3 D835, DNMT3A R882 and NPM1 c.863_864ins) by using several PCR techniques and also next-generation sequencing (NGS) analysis were performed. Results: Cytogenetic analysis did not reveal structural or numerical chromosomal anomalies. The patient’s DNA showed no copy number changes or aberrations (CNAs) following the MLPA analysis. By using several molecular technologies we found four mutations: FLT3-ITD, FLT3 D835 (c.2504A>T, D835V), DNMT3A R882C, and NPM1 c.863_864insTCTG. Challenges, benefits, applications and the limitations of each molecular technique used for the investigation of the mentioned mutation, and not only, are also described. Conclusion: All these techniques can be useful in the diagnosis of AML patients, each of them covering the limits of the other technique. New strategies for a positive, fast, accurate and reliable diagnosis are mandatory in cases with AML.

Clinical Evaluation of a Custom Genome Wide 44K Oligoarray for Copy Number Changes in Acute Myeloid Leukemia

Blood ◽  
2008 ◽  
Vol 112 (11) ◽  
pp. 4869-4869
Author(s):  
Ankita Patel ◽  
Rui-Yu Wang ◽  
Patricia Hixson ◽  
Jian Li ◽  
Aleksandar Milosavljevic ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Cytogenetic Analysis ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Repetitive Sequences ◽  
Chromosome Analysis ◽  
Chromosome 8 ◽  
Monosomy 7 ◽  
Copy Number Changes ◽  
Acute Myeloid

Abstract Oligonucleotide-based array CGH (aCGH) technique enables detection of submicroscopic copy number changes in cancer. This technique offers the flexibility to increase robustness by selecting best performing probes maximize the gene coverage while excluding repetitive sequences through a combination of bioinformatics and computation and increased resolution that allows for detection of smaller region of change. We designed a high resolution (44,000 probes) custom array using an Agilent platform and specifically targeted ~500 genes implicated in leukemogenesis. The disease gene regions have an average spatial resolution of ~1 oligo per 7.5 kb whereas the backbone regions have an average resolution of 1 oligo per 78 kb. A total of 13 samples from patients with AML have been studied to date. aCGH was performed in a blinded fashion and the results were compared to the results from routine cytogenetic analysis. A GAIN in copy number on the long arm of chromosome 8 at band 8q22.2 encompassing 2.05 Mb was observed in one of 7 cases with normal chromosome analysis. Of the remaining 6 cases, the results from aCGH were concordant with the abnormality detected by chromosome analysis in three although the size of the copy number change was determined with greater precision by aCGH. Additional copy number changes were observed in the remaining two cases. In one case, in addition to the deletion of 5q14q34 detected by cytogenetic analysis, a loss in copy number on the long arm of 17q at band 17q11.2 encompassing 1.8 Mb including the NF-1 gene was detected by aCGH. Two additional copy number changes in 3q26.1 and 8q24.1 were observed in another case in which monosomy 7 was observed cytogenetically. aCGH failed to detect a clone (2 cells) with a deletion on 5 from band 5p13q13 in one case. In total, 30% of the patients with chromosome analysis displayed an additional cryptic change by aCGH. Our results suggest that a subset of potentially significant genomic alteration may be missed by the conventional chromosome analysis. This pilot study demonstrates that aCGH offers high sensitivity and specificity for routine screening of copy number changes in acute myeloid leukemia.

DNA Copy Number Changes and Immunophenotype Pattern in Karyotypically Normal Acute Myeloid Leukemia Patients from an Indian Population

2012 ◽  
Vol 16 (4) ◽  
pp. 265-270 ◽  
Author(s):  
Nikesh Kawankar ◽  
Seema Korgaonkar ◽  
Lily Kerketta ◽  
Manisha Madkaikar ◽  
Farah Jijina ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Indian Population ◽  
Dna Copy Number ◽  
Copy Number Changes ◽  
Acute Myeloid

Genome-Wide Micro-Deletions and Amplifications Acquired at Relapse in Acute Myeloid Leukemia.

Blood ◽  
2009 ◽  
Vol 114 (22) ◽  
pp. 166-166 ◽  
Author(s):  
Manoj Raghavan ◽  
Manu Gupta ◽  
Tracy Chaplin ◽  
Sabah Khalid ◽  
T. Andrew Lister ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Conflicts Of Interest ◽  
Snp Array ◽  
Uniparental Disomy ◽  
Normal Karyotype ◽  
P Value ◽  
Copy Number Changes ◽  
Acute Myeloid

Abstract Abstract 166 Recurrence of acute myeloid leukemia AML has a poor prognosis with only 20% of adults surviving to 5 years. Therefore it is of importance to identify molecular changes that explain the pathogenesis of relapsed AML. Previous studies had not identified consistently acquired cytogenetic changes at relapse. Recently, acquired uniparental disomy due to mitotic recombination was described in 40% of relapsed AML (Raghavan et al 2008). Most of the events lead to homozygosity for FLT3 mutations. This study aimed to discover if there are further genetic abnormalities acquired at disease recurrence that cannot be identified by conventional cytogenetics, i.e. microdeletions or gains. Twenty-one presentation and relapse paired AML patient blood and marrow samples were stored with consent at St Bartholomew's Hospital, London. Eleven patient samples had a normal karyotype at diagnosis, two had favourable prognosis cytogenetics (inv(16) and t(8;21)) and others had varying numerical cytogenetic abnormalities and rearrangements associated with an intermediate prognosis. DNA from the samples was analysed by array based high-resolution single nucleotide polymorphism (SNP) genotyping (Affymetrix Human SNP array 6.0). Data was analysed using Partek Genome Browser (Partek, MO). In all cases, the leukemia infiltrate of the marrow or blood was greater than 60% and most cases were greater than 90% allowing accurate identification of DNA copy number changes. Abnormalities of a size that would be identified by cytogenetics were disregarded. Using segmentation analysis using a p-value less than 0.001, over 400 microdeletions and gains were detected that were acquired at relapse in the 21 pairs. Each of the copy number changes was less than 2 megabases in size. One AML sample with a normal karyotype at diagnosis and trisomy 8 and add(9)(q34) at relapse had not acquired any microdeletions or gains. In contrast, in other samples as many as 69 microdeletions/gains were detected. There was no correlation between increased complexity of the karyotype of the leukemia and the number of microdeletions/gains. Several of the acquired microdeletions/gains were in regions containing genes known to be involved in AML, including a deletion of 234Kb at 13q12.2 involving FLT3 and CDX2, and an acquired deletion at 21p11.2 of 150Kb involving exons encoding the runt domain of RUNX1. Another copy number gain was detected at the MLL locus, suggestive of partial tandem duplication. Other detected locations are in Table 1.Table 1Location by cytobandCopy number changeSize / KbP valueGene13q12.2Deletion23410−33FLT3, CDX221q22.12Deletion15010−13RUNX111q23.3Gain5.10.0099MLL11p15.4Gain830.00001NUP9817q21.31Deletion8.00.0007BRCA1The results indicate that recurrent AML may be associated with the deletion or gain of several genes involved in leukaemogenesis. Many other locations are involved throughout the genome, suggesting at least some of these are also involved in the clonal evolution of the leukaemia at recurrence. Further studies should identify novel genes from these regions involved in the pathogenesis of AML. Disclosures: No relevant conflicts of interest to declare.

Molecular Characterization of Chromosomal Abnormalities In Myelodysplastic Syndrome and Acute Myeloid Leukemia: Validation of An MLPA Protocol and Analysis Method for Use In a Diagnostic Setting

Blood ◽  
2010 ◽  
Vol 116 (21) ◽  
pp. 4849-4849
Author(s):  
Amber C. Donahue ◽  
Adam Abdool ◽  
Jay G Wohlgemuth ◽  
Chen-Hsiung Yeh
Keyword(s):  
Acute Myeloid Leukemia ◽  
Data Analysis ◽  
Myelodysplastic Syndrome ◽  
Copy Number ◽  
Myeloid Leukemia ◽  
Chromosomal Abnormalities ◽  
Analysis Method ◽  
Normal Range ◽  
Copy Number Changes ◽  
Acute Myeloid

Abstract Abstract 4849 Introduction: Current diagnostic screening strategies for copy number variations (CNVs) in myelodysplastic syndrome (MDS) and acute myeloid leukemia (AML) include fluorescence in situ hybridization (FISH) or karyotyping, both of which are time-consuming, costly, laborious, and lacking in resolution. Multiplex ligation-dependent probe amplification (MLPA) can be used to detect copy number changes in multiple loci simultaneously in a single PCR reaction, and boasts a resolution down to single exons. To adapt MLPA for use in routine clinical diagnostics, we have developed and validated a protocol for automatic data analysis and interpretation of common chromosomal abnormalities in MDS/AML. Patients and Methods: The study used a training set of 45 healthy subjects to establish a normal reference range for each individual probe. Using these ranges we built an automated Excel spreadsheet-based analysis system, which included multiple quality checks, and flagged samples failing these quality controls. Each probe was given a call of “no mutation detected,” “deletion,” or “gain,” based on whether the normalized ratio fell within or outside of the empirically-determined normal range for that probe. We then analyzed over 100 leukemia cases tested by FISH, including both suspected myeloid leukemia samples and suspected chronic lymphocytic leukemia (CLL) samples. Documented chromosomal abnormalities in CLL include 11q-, 17p- (loss of TP53), and trisomy 12, all of which had the potential to be detected by the probes in the MDS MLPA probemix. The greater prevalence of CLL and its associated CNVs provided additional positive controls for the validation of the MDS MLPA probemix and our analysis method. Results: The empirically-determined normal ranges demonstrated that some probes varied widely (3 standard deviation [3SD] normal range of 0.46–1.54), while others were extremely reliable (3SD normal range of 0.84–1.16). The MLPA assay demonstrated excellent overall accuracy (>90%) and specificity (>93%) for both suspected myeloid and CLL samples when compared to FISH. The sensitivity of the MLPA assay is somewhat lower than that of FISH, requiring a probe-dependent 20–40% positivity for a given CNV to be detected. However in several cases, the MDS MLPA assay was able to detect additional lesions too small to be seen by FISH. Conclusions: For MLPA, the total process-to-report time, including data analysis, is 2–3 days, versus the 7–10 days required for FISH analysis. In addition, the MLPA assay is substantially cheaper and considerably less labor-intensive than FISH. Our improved MLPA assay protocol and analysis method provides a clinically robust, multiplexed, high-throughput, high-resolution, and low-cost solution for detection of copy number changes in MDS/AML, and can therefore be used as a first-line screening test in a clinical laboratory. Disclosures: Donahue: Quest Diagnostics Inc.: Employment. Abdool: Quest Diagnostics Inc.: Employment. Wohlgemuth: Quest Diagnostics Inc.: Employment. Yeh: Quest Diagnostics Inc.: Employment.

scholarly journals Mate pair sequencing improves detection of genomic abnormalities in acute myeloid leukemia

2018 ◽  
Vol 102 (1) ◽  
pp. 87-96 ◽  
Author(s):  
Umut Aypar ◽  
Stephanie A. Smoley ◽  
Beth A. Pitel ◽  
Kathryn E. Pearce ◽  
Roman M. Zenka ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
Myeloid Leukemia ◽  
Mate Pair ◽  
Acute Myeloid

scholarly journals EVI1 and GATA2 misexpression induced by inv(3)(q21q26) contribute to megakaryocyte-lineage skewing and leukemogenesis

2020 ◽  
Vol 4 (8) ◽  
pp. 1722-1736 ◽  
Author(s):  
Ayaka Yamaoka ◽  
Mikiko Suzuki ◽  
Saori Katayama ◽  
Daiki Orihara ◽  
James Douglas Engel ◽  
...  
Keyword(s):  
Acute Myeloid Leukemia ◽  
High Risk ◽  
Myeloid Leukemia ◽  
Chromosomal Rearrangements ◽  
Leukemic Cells ◽  
Heterozygous Deletion ◽  
Hematopoietic Stem ◽  
Cellular Origin ◽  
A Cell ◽  
Acute Myeloid

Abstract Chromosomal rearrangements between 3q21 and 3q26 elicit high-risk acute myeloid leukemia (AML), which is often associated with elevated platelet and megakaryocyte (Mk) numbers. The 3q rearrangements reposition a GATA2 enhancer near the EVI1 (or MECOM) locus, which results in both EVI1 overexpression and GATA2 haploinsufficiency. However, the mechanisms explaining how the misexpression of these 2 genes individually contribute to leukemogenesis are unknown. To clarify the characteristics of differentiation defects caused by EVI1 and GATA2 misexpression and to identify the cellular origin of leukemic cells, we generated a system to monitor both inv(3) allele-driven EVI1 and Gata2 expression in 3q-rearranged AML model mice. A cell population in which both EVI1 and Gata2 were highly induced appeared in the bone marrows before the onset of frank leukemia. This population had acquired serial colony-forming potential. Because hematopoietic stem/progenitor cells (HSPCs) and Mks were enriched in this peculiar population, we analyzed the independent EVI1 and GATA2 contributions to HSPC and Mk. We found that inv(3)-driven EVI1 promotes accumulation of Mk-biased and myeloid-biased progenitors, Mks, and platelets, and that Gata2 heterozygous deletion enhanced Mk-lineage skewing of EVI1-expressing progenitors. Notably, inv(3)-directed EVI1 expression and Gata2 haploinsufficient expression cooperatively provoke a leukemia characterized by abundant Mks and platelets. These hematological features of the mouse model phenocopy those observed in human 3q AML. On the basis of these results, we conclude that inv(3)-driven EVI1 expression in HSPCs and Mks collaborates with Gata2 haploinsufficiency to provoke Mk-lineage skewing and leukemogenesis with excessive platelets, thus mimicking an important feature of human AML.

Sign in / Sign up

Export Citation Format

Share Document