Kategorie: Onkologická diagnostika
Téma: Cytogenetics and molecular diagnostics
Číslo abstraktu: P953
Autoři: Tereza Jančušková; Mgr. Radek Plachý, Ph.D.; Jiří Štika; Lucie Zemánková; David Warren Hardekopf; Dr. Thomas Liehr; Nadezda Kosyakova; Lenka Zejsková; Lucie Sedláčková; RNDr. Radek Čmejla, Ph.D.; MUDr. Soňa Peková (Chambon), Ph.D.
Acute leukemias (AL) comprise a heterogeneous group of hematologic malignancies, and individual patient responses to treatment can be difficult to predict. Monitoring of minimal residual disease (MRD) is thus very important and holds great potential for improving treatment strategies. Common MRD targets include recurrent cytogenetic abnormalities and mutations in important hematological genes; unfortunately well-characterized targets are lacking in many AL patients.
Our aim was to develop a flexible strategy for mapping of cytogenetically identified unique clone-specific abnormalities down to the single nucleotide level and, based on the sequence, design a specific real-time PCR assay for MRD assessment in AL patients without any previously described MRD marker.
Using a combination of molecular cytogenetic techniques (mFISH, mBAND) and molecular biological techniques (next-generation sequencing, long-range PCR, Sanger sequencing) we were able to characterize the DNA sequence flanking unique chromosomal breakpoints. For precise identification of these breakpoints we used fine-needle microdissection of derivative chromosomes followed by next-generation sequencing of the dissected material. Finally, we designed a specific real-time PCR assays for monitoring MRD level during the patients’ treatment. To test the applicability of the described approach, a real-time PCR assay based on a unique breakpoint from one patient was compared to quantification based on a well-characterized MRD target present in the same patient.
In conjunction with our previous proof of principle study on the K562 cell line presented at the EHA meeting in 2012, we mapped derivative chromosomes in 4 eligible AL patients [Patient 1 – der(4), der(11); patient 2 – der(10), der(11); patient 3 – der(8); patient 4 – der(7)] and performed real-time PCR quantification of unique MRD markers for each patient. A comparison of these newly-designed assays to a standard assay used in clinical practice shows that our technical approach is suitable for the identification of new molecular MRD markers in AL patients.
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Summary / Conclusion:
The combination of cytogenetic and molecular methods described here enabled us to proceed from the chromosomal level (cytogenetically identified unique marker) to the molecular level (unique DNA sequence). Using this procedure, many chromosomal translocations can be used to identify a target for detecting and quantifying MRD. Our work clearly shows that moving from the chromosomal level to the nucleotide level is feasible and readily applicable for eligible AL patients. The time required for the entire procedure under reasonable standard conditions, from receiving the diagnostic sample to developing the real-time MRD assay, is approximately up to six weeks, allowing its use in clinical practice as a tool for personalized medicine in hemato-oncology.
Keywords: Acute leukemia, Cytogenetics, Minimal residual disease (MRD), Molecular markers
Datum přednesení příspěvku: 15. 6. 2013