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E.K.M. Mack et al.
neous populations. For example, gain of chromosome 19 has been reported to be missed frequently by convention- al banding techniques,48 as observed here in patient AML- 28. Nevertheless, we did not verify this finding by an alternative detection method. Given that NGS technolo- gy is increasingly used by diagnostic laboratories, the lc- WGS strategy described here opens up the possibility of performing high-resolution numerical karyotyping of AML samples on a routine basis. On the other hand, unlike classical chromosome banding or FISH analyses, CNV karyotyping combined with targeted resequencing of chromosomal fusions will not identify balanced translocations that are not covered by the sequencing panel. These features include some abnormalities defin- ing AML with myelodysplasia-related changes, which is not, however, exclusively a genetic diagnosis.9 Hence, larger cohorts of AML patients will have to be examined in order to develop a new risk stratification system that incorporates the gain of information obtained by lc-WGS (or array-based comparative genomic hybridization) compared to cytogenetics and potential gaps of knowl- edge that may emerge with a particular CNV karyotyping strategy. Future studies also need to include more exten- sive validation of the sensitivity of our CNV karyotyping
assay to detect chromosomal gains and losses in regions that have not been specifically investigated here.
In summary, to the best of our knowledge, our work represents the fastest and most comprehensive analysis platform for diagnosing AML developed so far. Combining molecular genetics and cytogenetics in one NGS run will pave the way for differentiated manage- ment of AML patients not only in clinical trials, but also in a standard-of-care setting, as is currently emerging with midostaurin as the first targeted agent in induction therapy for patients with FLT3 alterations.49
Funding
This work was supported by grants from Rhön Klinikum AG (RKA n. 64 to EKMM, CB, MGK), Deutsche Forschungsgemeinschaft KFO210 (BR2232/2 to CB; Ne310/18- 1 to AN), Deutsche José Carreras Leukämie-Stiftung (AH06-01 to AN, 18R/2016 to EKMM) and Stiftung PE Kempkes (06/2014 to EKMM).
Acknowledgments
We thank Lisa-Marie Koch, Ute Niebergall and Kathleen Stabla for excellent technical assistance and Prof. Edison T. Liu for helpful advice.
References
1. Ley TJ, Miller C, Ding L, et al. Genomic and epigenomic landscapes of adult de novo acute myeloid leukemia. N Engl J Med. 2013;368(22):2059–2074.
2. Dohner H, Estey E, Grimwade D, et al. Diagnosis and management of AML in adults: 2017 ELN recommendations from an international expert panel. Blood. 2017;129 (4):424-447.
3. Papaemmanuil E, Gerstung M, Bullinger L, et al. Genomic classification and prognosis in acute myeloid leukemia. N Engl J Med. 2016;374(23):2209–2221.
4. Dohner H, Weisdorf DJ, Bloomfield CD. Acute myeloid leukemia. N Engl J Med. 2015;373(12):1136–1152.
5. Grimwade D, Ivey A, Huntly BJP, et al. Molecular landscape of acute myeloid leukemia in younger adults and its clinical relevance. Blood. 2016;127(1):29–41.
6. Lo-Coco F, Avvisati G, Vignetti M, et al. Retinoic acid and arsenic trioxide for acute promyelocytic leukemia. N Engl J Med. 2013;369(2):111–121.
7. AuCH,WaA,HoDN,ChanTL,MaES. Clinical evaluation of panel testing by next- generation sequencing (NGS) for gene muta- tions in myeloid neoplasms. Diagn Pathol. 2016;11:11.
8. Xu C, Nezami Ranjbar MR, Wu Z, DiCarlo J, Wang Y. Detecting very low allele fraction variants using targeted DNA sequencing and a novel molecular barcode-aware variant caller. BMC Genomics. 2017;18(1):5.
9. Arber DA, Orazi A, Hasserjian R, et al. The 2016 revision to the World Health Organization classification of myeloid neo- plasms and acute leukemia. Blood. 2016;127 (20):2391–2405.
10. Zheng Z, Liebers M, Zhelyazkova B, et al. Anchored multiplex PCR for targeted next- generation sequencing. Nat Med. 2014;20(12):1479–1484.
11. Liu S, Song L, Cram DS, et al. Traditional karyotyping vs copy number variation sequencing for detection of chromosomal abnormalities associated with spontaneous miscarriage. Ultrasound Obstet Gynecol. 2015;46(4):472–477.
12. Heitzer E, Ulz P, Belic J, et al. Tumor-associ- ated copy number changes in the circulation of patients with prostate cancer identified through whole-genome sequencing. Genome Med. 2013;5(4):30.
13. Hagenkord JM, Parwani AV, Lyons-Weiler MA, et al. Virtual karyotyping with SNP microarrays reduces uncertainty in the diag- nosis of renal epithelial tumors. Diagn Pathol. 2008;3:44.
14. Wang T-L, Maierhofer C, Speicher MR, et al. Digital karyotyping. Proc Natl Acad Sci U S A 2002;99(25):16156–16161.
15. Pirooznia M, Goes FS, Zandi PP. Whole- genome CNV analysis: advances in compu- tational approaches. Front Genet. 2015;6:138.
16. Xie C, Tammi MT. CNV-seq, a new method to detect copy number variation using high- throughput sequencing. BMC Bioinformatics. 2009;10:80.
17. Li X, Chen S, Xie W, et al. PSCC: sensitive and reliable population-scale copy number variation detection method based on low coverage sequencing. PLoS One. 2014;9(1):e85096.
18. Puttmann S, Senner V, Braune S, et al. Establishment of a benign meningioma cell line by hTERT-mediated immortalization. Lab Invest. 2005;85(9):1163–1171.
19. Gallagher R, Collins S, Trujillo J, et al. Characterization of the continuous, differen- tiating myeloid cell line (HL-60) from a patient with acute promyelocytic leukemia. Blood. 1979;54(3):713-733.
20. Karnan S, Tsuzuki S, Kiyoi H, et al. Genomewide array-based comparative genomic hybridization analysis of acute promyelocytic leukemia. Genes
Chromosom Cancer. 2006;45(4):420–425. 21. Lanotte M, Martin-Thouvenin V, Najman S, Balerini P, Valensi F, Berger R. NB4, a matu- ration inducible cell line with t(15;17) mark- er isolated from a human acute promyelo- cytic leukemia (M3). Blood. 1991;77(5):1080-
1086.
22. Shipley J, Weber-Hall S, Birdsall S. Loss of
the chromosomal region 5q11-q31 in the myeloid cell line HL-60: characterization by comparative genomic hybridization and flu- orescence in situ hybridization. Genes Chromosom Cancer. 1996;15(3):182–186.
23. Mozziconacci MJ, Rosenauer A, Restouin A, et al. Molecular cytogenetics of the acute promyelocytic leukemia-derived cell line NB4 and of four all-trans retinoic acid-resis- tant subclones. Genes Chromosom Cancer. 2002;35(3):261–270.
24. Liang JC, Ning Y, Wang RY, et al. Spectral karyotypic study of the HL-60 cell line: detection of complex rearrangements involving chromosomes 5, 7, and 16 and delineation of critical region of deletion on 5q31.1. Cancer Genet Cytogenet. 1999;113(2):105–109.
25. Collins S, Groudine M. Amplification of endogenous myc-related DNA sequences in a human myeloid leukaemia cell line. Nature. 1982;298(5875):679–681.
26. Von Hoff DD, Forseth B, Clare CN, Hansen KL, VanDevanter D. Double minutes arise from circular extrachromosomal DNA inter- mediates which integrate into chromosomal sites in human HL-60 leukemia cells. J Clin Invest. 1990;85(6):1887–1895.
27. Hoffmann JC, Stabla K, Burchert A, et al. Monitoring of acute myeloid leukemia patients after allogeneic stem cell transplan- tation employing semi-automated CD34+ donor cell chimerism analysis. Ann Hematol 2014;93(2):279–285.
28. Luthra R, Patel KP, Reddy NG, et al. Next- generation sequencing-based multigene mutational screening for acute myeloid
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