scholarly journals Application of RNA Sequencing Detects a Large Number of Novel Fusion Transcripts in Patients with AML and MDS

Blood ◽  
2019 ◽  
Vol 134 (Supplement_1) ◽  
pp. 887-887 ◽  
Author(s):  
Anna Stengel ◽  
Rabia Shahswar ◽  
Wencke Walter ◽  
Manja Meggendorfer ◽  
Wolfgang Kern ◽  
...  
Keyword(s):  
Rna Sequencing ◽  
High Frequency ◽  
Hematological Malignancies ◽  
Gene Fusions ◽  
Very High Frequency ◽  
Employment Equity ◽  
Tp53 Mutations ◽  
Fusion Transcripts ◽  
Equity Ownership ◽  
Very High

Background: Chimeric transcripts are frequent genetic abnormalities in hematological malignancies that often contribute to leukemogenesis and play crucial roles for risk stratification, MRD monitoring and targeted therapy. However, current standard techniques for detection of gene fusions (chromosome banding analysis (CBA) and fluorescence in situ hydridization (FISH)) are only able to predict known and/or non-cryptic gene fusions. RNA sequencing (RNAseq) has successfully been applied for determination of genomic gene fusions. Aim: Comprehensive analysis of fusion genes in AML (acute myeloid leukemia) and MDS (myelodysplastic syndrome) patients and evaluation of the beneficial use of RNAseq in detecting recurrent and novel fusions. Methods: RNAseq was performed for 579 AML and 630 MDS patients for the detection of recurrent and novel fusion transcripts. WGS (whole genome sequencing) and cytogenetics (CBA, FISH) were applied for validation of the respective transcripts. 151 bp paired-end reads were produced on a NovaSeq 6000 system (Illumina, San Diego, CA) with a yield between 35 and 125 million paired reads per sample. Reciprocal fusion transcripts were counted as one fusion event. All reported p-values are two-sided and were considered significant at p<0.05. Results: After stringent filtering and validation by WGS and/or cytogenetics, for AML, 279 fusion events (corresponding to 147 unique gene fusions) were detected in 208 cases (36% of patients). 213/279 (76%) were confirmed by WGS and cytogenetics, 54/279 cases (19%) by WGS only and 12/279 (4%) cases by cytogenetics only. As a proof of principle, entity-defining rearrangements were detected most frequently: RUNX1-RUNX1T1 (n=42), CBFB-MYH11 (n=39) and PML-RARA (n=39). Other recurrent fusions included KMT2A-MLLT3 (n=6), KMT2A-MLLT10 (n=3) and DEK-NUP214 (n=4) (Fig 1A). However, in addition a high number (130/279, 47%) of so far unreported gene fusions were detected, including 49/130 inter-chromosomal and 81/130 intra-chromosomal fusions. These comprised 92/130 cases for which both fusion partners have not been reported before and 38/130 cases with a novel partner of a gene previously reported in hematological malignancies, including novel partners for NUP98 (XRN1), ETV6 (FAAP100, ARNTL2, SMCO2), RUNX1 (THOC6, EIF3E, OPHN1, TMEM50B), RARA (CCDC33), CBFB (TTC3, HMGB1) and KMT2A (NCBP1, ARHGEF12) (Fig 1B) (all validated by WGS and/or cytogenetics (see above)). Most of the novel fusions were detected only once, however two of them were observed in two patients each (NRIP2-ITFG2, CTDSP1-CFLAR). Moreover, cases with novel fusions were characterized by a very high frequency of TP53 mutations (59% vs. 1% in cases with known fusions and 9% in cases with no detected fusions, p<0.001), whereas FLT3-ITD and NPM1 mutations were rather rare (NPM1: 8% (novel) vs. 0% (known) vs. 35% (no fusion); FLT3-ITD: 3% (novel) vs. 14% (known) vs. 24% (no fusion). Consequently, a large number of cases with novel fusions depicted a complex karyotype (62% (novel) vs. 1% (known) vs. 9% (no fusion)). Regarding age, patients with known fusions were significantly younger than patients with novel fusions or without detected fusions (median age: 54 years (known) vs. 72 (novel) vs. 70 (no fusion), p<0.001). For MDS, 30 fusions (29 unique transcripts) were observed in 28/630 cases (4% of all patients). 4/30 (13%) of detected gene fusions were inter-chromosomal, while the majority (26/30, 87%) was intra-chromosomal. 27/30 (90%) fusions were validated by WGS, 9/30 (30%) by CBA, comprising 6/30 (20%) cases that were validated by both methods. Only one of the detected fusions was detected in >1 patient (n=2) and was described previously (MECOM-NRIP1), all others (n=28) are so far unreported. Of these, 23/28 comprise both so far undescribed fusion partners, whereas for 5/28 fusions one partner was previously reported to function in hematological neoplasms (e.g. MYB-PEX7, RABEP1-NUP88). Conclusions: (1) A large number of novel fusions were detected by RNAseq and validated by WGS and cytogenetics, especially in AML. (2) These novel fusions correlate with a very high frequency of TP53 mutations, their pathogenic role has to be evaluated further. (3) This data may provide the basis for identifying potential new actionable targets (e.g. for personalized vaccine or adoptive cell-based therapy development) and developing markers for patient sensitive MRD monitoring. Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Walter:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

scholarly journals Comparison of TP53 Alterations in Hematological Malignancies

Blood ◽  
2015 ◽  
Vol 126 (23) ◽  
pp. 4819-4819
Author(s):  
Anna Stengel ◽  
Wolfgang Kern ◽  
Torsten Haferlach ◽  
Susanne Schnittger ◽  
Melanie Zenger ◽  
...  
Keyword(s):  
Burkitt Lymphoma ◽  
Tp53 Mutation ◽  
Hematological Malignancies ◽  
Complex Karyotype ◽  
Mutation Load ◽  
Employment Equity ◽  
Copy Number State ◽  
Tp53 Mutations ◽  
Cytogenetic Aberrations ◽  
Equity Ownership

Abstract Background: TP53 is altered in ~50% of human cancers. Alterations include mutations and deletions. Both frequently occur together, supporting the classical "two-hit" hypothesis for tumor-suppressor genes. Aim: Comparison of TP53 mutation/deletion patterns in different hematological malignancies, including AML, MDS, ALL, Burkitt lymphoma, CLL and T-PLL. We analyzed (i) the frequencies of TP53 mutations and deletions, (ii) the types of mutation, (iii) the mutation load, (iv) the correlations to cytogenetic aberrations, (v) the age dependency, and (vi) impact on survival. Patient cohort and methods: A total of 3383 cases (AML: n=858, MDS: n=943, ALL: n=358, Burkitt lymphoma: n=25, CLL: n=1148 and T-PLL: n=51) were analyzed for TP53 deletions by interphase FISH determining the copy number state and for TP53 mutations by next-generation amplicon deep sequencing. Karyotype data was available for all cases. Results: Overall, alterations in TP53 were detected in 361/3383 cases (11%; 186 cases with mutation only (mut only), 51 cases with deletion only (del only), 124 cases with mutation and deletion (mut+del)). Regarding the respective entities, the highest frequency of TP53 alterations was observed in patients with Burkitt lymphoma (total alteration frequency: 56%, mut+del: 12%, mut only: 44%, no case del only). Alterations in TP53 also occured with a high incidence in patients with T-PLL (total: 30%; mut+del: 10%; mut only: 4%; del only: 16%) followed by cases with ALL (total: 19%; mut+del: 6%; mut only: 8%; del only: 5%) and AML (total: 13%; mut+del: 5%; mut only: 7%; del only: 1%). By contrast, TP53 alterations occurred less frequently in patients with CLL (total: 8%; mut+del: 4%; mut only: 3%; del only: 1%) and MDS (total: 7%; mut+del: 1%; mut only: 5%; del only: 1%). Missense mutations were found to be the most abundant mutation type in all entities analyzed with a frequency ranging from 71% - 88%. In all entities mainly one mutation per case was detected; however, MDS cases were found to harbour a statistically increased proportion of cases with two mutations compared to the other entities (p = 0.003). High TP53 mutation loads were detected in T-PLL (median: 88%) and AML (47%), whereas the lower ones were found in ALL (28%), Burkitt lymphoma (39%), MDS (39%), and CLL (36%). A strong correlation of TP53 alterations with a complex karyotype was observed in AML (of patients with TP53 alteration: 5% with normal karyotype, 67% with complex karyotype, 28% with other aberrations), ALL (16% normal, 45% complex, 39% other), MDS (14% normal, 53% complex, 33% other), and T-PLL (20% normal, 47% complex, 33% other). By contrast, in CLL and Burkitt lymphoma, TP53 alterations were mainly correlated with other aberrations (CLL: 10% normal, 30% complex, 60% other; Burkitt: 29% normal, 0% complex, 71% other). TP53 mut and TP53 mut+del were significantly more frequent in patients ≥ 60 vs < 60 years in AML (9% vs. 2% for mut only, p < 0.001; 7% vs. 2% for mut+del, p = 0.001) and ALL (12% vs. 6% for mut only, p < 0.001; 13% vs. 3% for mut+del, p = 0.001). By contrast, no such differences were observed for patients with CLL, MDS, T-PLL and Burkitt lymphoma. Moreover, TP53 alterations (especially of TP53 mut+del) had a significant negative impact on OS in all entities except for T-PLL and Burkitt lymphoma, most probably due to their overall short OS or due the lower number of cases. Conclusion: The frequency of TP53 mutations and/or deletions as well as the mutation load clearly varied between different hematological malignancies with the highest incidence of TP53 mut in patients with Burkitt lymphoma (56%) and a rather low frequency in CLL (7%) and MDS (6%). TP53 del were frequent in patients with T-PLL (26%) and Burkitt lymphoma (12%) and are hardly found in MDS cases (2%). TP53 alterations are correlated to higher age in AML and ALL. Moreover, alterations in TP53 are correlated to a short OS and to a complex karyotype, with the exception of Burkitt lymphoma and CLL, were they were found to be associated to other cytogenetic aberrations. Thus, TP53 mutations and deletions need further investigation in the future, especially regarding their clinical impact in different hematologic entities. Disclosures Stengel: MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Schnittger:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Zenger:MLL Munich Leukemia Laboratory: Employment. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Targeted RNA Sequencing Is Capable of Identifying Thus Far Unknown Partner Genes in Leukemias with Rare Translocations and Provides Important Clinical and Therapeutical Information

Blood ◽  
2016 ◽  
Vol 128 (22) ◽  
pp. 2857-2857
Author(s):  
Claudia Haferlach ◽  
Niroshan Nadarajah ◽  
Manja Meggendorfer ◽  
Nadine Dicht ◽  
Anna Stengel ◽  
...  
Keyword(s):  
Precision Medicine ◽  
Rna Sequencing ◽  
San Diego ◽  
Chromosome Banding ◽  
Gene Fusions ◽  
Rt Pcr ◽  
Employment Equity ◽  
Banding Analysis ◽  
Chromosome Banding Analysis ◽  
Equity Ownership

Abstract Background: The genomic landscape of hematological malignancies has been resolved mainly based on whole exome and whole genome sequencing, primarily targeting gene mutations. Beside mutations also gene fusions function as therapeutic targets, impressively shown for e.g. BCR-ABL1 and ETV6-PDGFRB. Hence, the need for a comprehensive genetic analysis is increasing, as it is the basis for precision medicine, selecting treatment based on genotype and providing markers for disease monitoring. Aim: To test the value of targeted RNA sequencing in a routine diagnostic work up. Patients and Methods: 38 cases were selected in which rearrangements involving KMT2A (n=8), RUNX1 (n=19), ETV6 (n=9), RARA (n=1) and JAK2 (n=1) had been identified by chromosome banding analysis (CBA) complemented by FISH analysis. In all cases the partner gene could not be identified using standard methods. Targeted RNA sequencing was performed using the TruSight RNA Fusion panel (Illumina, San Diego, CA) consisting of 7690 probes covering 507 genes known to be involved in gene fusions. Library was prepared according to manufacturer's protocol with ~50ng DNA extracted from fresh/frozen samples. This assay allows the capture of all targeted transcripts. Sequencing was performed on two NextSeq runs (Illumina, San Diego, CA) with 20 multiplexed samples including two samples with known fusions as positive control samples. Analysis was performed with the RNA-Seq Alignment App (BaseSpace Sequence Hub) using Star for Alignment and Manta for gene fusion calling with default parameters (Illumina, San Diego, CA). Results: In 22/38 cases with rearrangements involving KMT2A (n=8), RUNX1 (n=8), ETV6 (n=4), RARA (n=1) or JAK2 (n=1) this approach led to important new information: The following partner genes for KMT2A were identified: MLLT10 (n=2), MLLT1 (n=2), ITPR2, FLNC, ASXL2 and ARHGEF12. MLLT10 and MLLT1 are two of the most frequent partner genes of KMT2A, while KMT2A-ARHGEF12 fusions are rare. Fusion of KMT2A to ITPR2, FLNC, or ASXL2 have not been reported yet. Seven different partner genes were identified in RUNX1 translocated cases. These were PLAG1 (n=2), PRDM16, MECOM, ZFPM2, MAN1A2, N6AMT2, and KIAA1549L. PRDM1, MECOM and ZFPM2 have previously been described in the literature as RUNX1 partner genes but were not suspected in our cases as partner genes due to complex cytogenetic rearrangements in CBA. The other identified partner genes have not been described so far. Interestingly, PRDM1, MECOM, ZFPM2 and the newly identified PLAG1 are all members of the C2H2-type zinc finger gene family. Four different partner genes were identified in ETV6 rearranged cases: ABL1, CCDC126, CLPTM1L, and CFLAR-AS1. Most strikingly was the identification of the ETV6-ABL1 fusion, which could not be suspected by cytogenetics as the 5' ETV6 FISH signal was located on chromosome 7. This ETV6-ABL1 fusion was confirmed by conventional RT-PCR. In an ALL patient a JAK2-PPFIBP1 fusion was identified leading to classification as a BCR-ABL1-like ALL. In an APL patient showing an ins(17;11)(q12;q14q23) in chromosome banding analysis a ZBTB16-RARA fusion was identified and thus resistance to all-trans retinoic acid, arsenic trioxide, and anthracyclines can be predicted. All these fusions were not detectable by our routine RT-PCR analyses as these assays cover only the most frequently occurring breakpoints in fusions with known partner genes, but might miss very rare variants. For all yet undescribed fusion partners routine assays are not available. Based on the results of targeted RNA sequencing quantitative PCR assays for MRD monitoring can now be established. In 11 cases with a RUNX1 rearrangement and 5 cases with an ETV6 rearrangement no fusion transcript was identified. Further analyses will have to clarify whether in these cases no transcript was derived from the genomic rearrangement. Conclusions: 1) Targeted RNA sequencing was able to identify and characterize rare gene fusions and thus provided the basis for the design of RT-PCR based assays for monitoring MRD. 2) Targetable genetic aberrations were identified, which were not identifiable by chromosome banding analysis but would now lead to more individualized treatment. 3) Thus, targeted RNA sequencing may be a valuable tool in routine diagnostics for patients with rearrangements unresolved by standard techniques, also paving the way to precision medicine in a considerable number of patients. Disclosures Haferlach: MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Nadarajah:MLL Munich Leukemia Laboratory: Employment. Meggendorfer:MLL Munich Leukemia Laboratory: Employment. Dicht:MLL Munich Leukemia Laboratory: Employment. Stengel:MLL Munich Leukemia Laboratory: Employment. Kern:MLL Munich Leukemia Laboratory: Employment, Equity Ownership. Haferlach:MLL Munich Leukemia Laboratory: Employment, Equity Ownership.

Experimental Investigation of very High Frequency Slot Antenna on M-60A-1 Tank

1977 ◽  
Author(s):  
D. V. Campbell ◽  
William Kennebeck ◽  
A. Zanella ◽  
Paul Sexton
Keyword(s):  
Experimental Investigation ◽  
High Frequency ◽  
Slot Antenna ◽  
Very High Frequency ◽  
Very High

A collision feedback based multiple access control protocol for very high frequency data exchange system in E-navigation

2021 ◽  
pp. 1-16
Author(s):  
Xu Hu ◽  
Bin Lin ◽  
Ping Wang ◽  
Hongguang Lyu ◽  
Tie-Shan Li
Keyword(s):  
Access Control ◽  
High Frequency ◽  
Multiple Access ◽  
Data Exchange ◽  
High Frequency Data ◽  
Frequency Data ◽  
Exchange System ◽  
Very High Frequency ◽  
Time Division ◽  
Very High

Abstract The very high frequency data exchange system (VDES) is promising in promoting electronic navigation (E-navigation) and improving navigation safety. The multiple access control (MAC) protocol is crucial to the transmission performance of VDES. The self-organising time division multiple access (SOTDMA) protocol, as the only access mode given by current recommendations, leads to a high rate of transmission collisions in the traditional automatic identification system (AIS), especially with heavy traffic loads. This paper proposes a novel feedback based time division multiple access (FBTDMA) protocol to address the problems caused by SOTDMA, such that collision of transmissions can be avoided in information transmission among vessels. Simulation results demonstrate that the proposed FBTDMA outperforms the traditional SOTDMA in terms of channel utilisation and throughput, and significantly reduces the transmission collision rate. The study is expected to provide insights into VDES standardisation and E-navigation modernisation.

scholarly journals Measurement of Electrical Properties of Superconducting YBCO Thin Films in the VHF Range

Materials ◽  
2021 ◽  
Vol 14 (12) ◽  
pp. 3360
Author(s):  
Yakir Dahan ◽  
Eldad Holdengreber ◽  
Elichai Glassner ◽  
Oz Sorkin ◽  
Shmuel E. Schacham ◽  
...  
Keyword(s):  
Thin Films ◽  
Theoretical Model ◽  
Quality Factor ◽  
High Frequency ◽  
Unit Length ◽  
Superconducting Thin Films ◽  
Electrical Parameters ◽  
Very High Frequency ◽  
Production Constraints ◽  
Very High

A new measurement technique of electrical parameters of superconducting thin films at the Very High Frequency (VHF) range is described, based on resonators with microstrip (MS) structures. The design of an optimal resonator was achieved, based on a thorough theoretical analysis, which is required for derivation of the exact configuration of the MS. A theoretical model is presented, from which an expression for the attenuation of a MS line can be derived. Accordingly, simulations were performed, and an optimal resonator for the VHF range was designed and implemented. Production constraints of YBa2Cu3O7 (YBCO) limited the diameter of the sapphire substrate to 3″. Therefore, a meander configuration was formed to fit the long λ/4 MS line on the wafer. By measuring the complex input reflection coefficients of a λ/4 resonator, we extracted the quality factor, which is mainly affected by the dielectric and conductor attenuations. The experimental results are well fitted by the theoretical model. The dielectric attenuation was calculated using the quasi-static analysis of the MS line. An identical copper resonator was produced and measured to compare the properties of the YBCO resonator in reference to the copper one. A quality factor of ~6·105 was calculated for the YBCO resonator, three orders of magnitude larger than that of the copper resonator. The attenuation per unit length of the YBCO layer was smaller by more than five orders of magnitude than that of the copper.

Da eine oder mehrere betroffen …

2020 ◽  
Vol 48 (1) ◽  
pp. 47-100
Author(s):  
Melitta Gillmann
Keyword(s):  
High Frequency ◽  
Formal Language ◽  
Subordinate Clause ◽  
Matrix Clause ◽  
Very High Frequency ◽  
Corpus Study ◽  
Causal Relations ◽  
Legal Texts ◽  
The Matrix ◽  
Very High

AbstractBased on a corpus study conducted using the GerManC corpus (1650–1800), the paper sketches the functional and sociosymbolic development of subordinate clause constructions introduced by the subjunctor da ‘since’ in different text genres. In the second half of the 17th and the first half of the 18th century, the da clauses were characterized by semantic vagueness: Besides temporal, spatial and causal relations, the subjunctor established conditional, concessive, and adversative links between clauses. The corpus study reveals that different genres are crucial to the readings of da clauses. Spatial and temporal usages, for example, occur more often in sermons than in other genres. The conditional reading, in contrast, strongly tends to occur in legal texts, where it displays very high frequency. This could be the reason why da clauses carry indexical meaning in contemporary German and are associated with formal language. Over the course of the 18th century, the causal usages increase in all genres. Surprisingly, these causal da clauses tend to be placed in front of the matrix clause despite the overall tendency of causal clauses to follow the matrix clause.

scholarly journals Sintering, Microstructure, and Dielectric Properties of Copper Borates for High Frequency LTCC Applications

Materials ◽  
2021 ◽  
Vol 14 (14) ◽  
pp. 4017
Author(s):  
Dorota Szwagierczak ◽  
Beata Synkiewicz-Musialska ◽  
Jan Kulawik ◽  
Norbert Pałka
Keyword(s):  
Dielectric Properties ◽  
Dielectric Permittivity ◽  
High Frequency ◽  
Ceramic Materials ◽  
Sintering Behavior ◽  
Terahertz Time Domain Spectroscopy ◽  
Very High Frequency ◽  
X Ray ◽  
Low Dielectric ◽  
Very High

New ceramic materials based on two copper borates, CuB2O4 and Cu3B2O6, were prepared via solid state synthesis and sintering, and characterized as promising candidates for low dielectric permittivity substrates for very high frequency circuits. The sintering behavior, composition, microstructure, and dielectric properties of the ceramics were investigated using a heating microscope, X-ray diffractometry, scanning electron microscopy, energy dispersive spectroscopy, and terahertz time domain spectroscopy. The studies revealed a low dielectric permittivity of 5.1–6.7 and low dielectric loss in the frequency range 0.14–0.7 THz. The copper borate-based materials, owing to a low sintering temperature of 900–960 °C, are suitable for LTCC (low temperature cofired ceramics) applications.

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