Číslo abstraktu: 0684
Autoři: MUDr. Soňa Peková (Chambon), Ph.D.; Tereza Jančušková; Mgr. Radek Plachý, Ph.D.; David Warren Hardekopf; Dr. Thomas Liehr; A. Weise; Nadezda Kosyakova; Jiří Štika; Lenka Zejsková; Lucie Sedláčková; Lucie Krutílková; Radek Čmejla, PhD
Background. Acute myeloid leukemias (AML) in adulthood represent a heterogeneous entity, characterized by a recurrent chromosomal/genetic abnormality in only 50% of cases. In the remaining individuals, no common molecular abnormality can be identified using standard diagnostic screening (AcutePlexX1, mutations in NPM1, WT1, CEBPa and others), though unique cytogenetic abnormalities can often be detected. As many AML patients are eligible for curative treatment, techniques allowing specific and sensitive minimal residual disease (MRD) monitoring are highly needed. Aims. To develop a technique that would allow mapping of cytogenetically identified unique clone-specific abnormalities from the chromosome level to the nucleotide level, enabling us to develop clone-specific quantitative Real-Time PCR assays for sensitive and specific MRD monitoring. Methods. Molecular-cytogenetic techniques (mFISH, mBAND, BAC-FISH), chromosome microdissection, next generation sequencing, long-range PCR and direct Sanger sequencing were used to map the chromosomal translocation in the der(10)t(3;10)(p21.3;q23) characteristic for the cell line K562. This model cell line was chosen to show the feasibility and reproducibility of the technique as a proof of principle, with prospective continuation to authentic patient samples. After cytogenetic identification of the chromosomal translocation (Figure 1A), the derivative chromosome was microdissected using a fine needle (Figure 1B). The microdissected fragments were directly subjected to whole genome amplification (WGA; Figure 1C) and then sequenced on the GS Junior platform for next generation sequencing (Figure 1D). Obtained reads were aligned to reference sequences of chromosomes 3 and 10, using in-house developed software (Figure 1E). The last mapped reads from both chromosomes were used as docking sites for primers for long-range PCR to amplify the putative breakpoint (Figure 1F). The long-range PCR products were directly sequenced using Sanger sequencing to reveal the precise nucleotide sequence of the breakpoint (Figure 1G). Results. Using a combination of cytogenetic and molecular approaches, we mapped the K562-unique translocation in der(10)t(3;10)(p21.3;q23) from the chromosomal level to the nucleotide level (Figure 1). Direct sequencing of this breakpoint revealed a head-to-head fusion of genes CDC25A and GRID1. The time demand of the whole procedure, starting from mFISH and ending with the actual sequence of the breakpoint was approximately 6 weeks. This is optimal timing for a standard clinical setting, when the laboratory receives the first followup samples one month after diagnosis. Summary and Conclusions. Current technologies of molecular cytogenetics and molecular biology open new vistas in the detection and identification of unique, clone-specific genetic abnormalities in patients with AML. As only half of the individuals with AML can be molecularly MRD followed-up using recurrent genetic abnormalities, there is still a sizeable proportion of patients who might benefit from the identification of the “finger prints”of their malignant cells for the design of clone-specific MRD Real-Time PCR assays. Our work clearly shows that “walking”from the chromosomal level to the nucleotide level is feasible and readily applicable for eligible AML patients. Acknowledgements. The work was supported by the Grant Agency of Ministry of Industry and Trade of the Czech Republic and in part by the Monika-Kutzner Foundation.
Reference Plachy R. et al., 2011; Blood, 118:1083, Abstract 2526.
Figure 1. The outline of the technique used to walk from the chromosome level to the nucleotide level.
Haematologica, 2012; 97(s1): 279
Datum přednesení příspěvku: 14. 6. 2012