interfáznífish-cloudfrontnet.ppt

A Novel Approach for Unique MRD Markers Identification in Acute Leukemia Patients Tereza Jan?u?ková synlab genetics s.r.o. Evropska 176/16, Prague, Czech Republic Departments: Cytogenetics Molecular hematooncology Molecular detection of pathogens Molecular detection of rare genetic syndromes Charles Bridge and Prague Castle synlab genetics, Laboratory for Molecular Diagnostics Czech Republic, Prague Introduction – Acute Leukemia Acute leukemias (AL) – acute myeloid leukemia (AML) and acute lymphoid leukemia (ALL) Different prognosis depending on many factors Sensitive minimal residual disease (MRD) monitoring: – strong prognostic factor – assessment of the quality of treatment response – prediction of individual risk of relapse Real-time PCR technique – sensitivity ~ 10-5 – molecular marker is necessary Introduction – Molecular Markers adult ALL patients – in majority cases suitable marker is identified IgH/TCR gene rearrangements cytogenetic abnormalities = fusion transcripts (BCR-ABL, MLL-AF4…) adult AML patients – suitable molecular marker in 50 % only cytogenetic abnormalities = fusion transcripts (PML-RARA, AML1-ETO…) gene mutation (NPM1, CEBPA, WT1, c-KIT…) Our Aim To develop a flexible strategy for identification of unique molecular targets for sensitive MRD assessment in AL patients - mapping of cytogenetic abnormalities down to the single nucleotide level - design a specific real-time PCR assay Study Design Pilot study - cell line K562 Patients with acute leukemia DOP-PCR Sanger sequencing CDC25A chromosome 3 GRID1 chromosome 10 Long-range PCR ~ 4,5 kb Next-generation sequencing and data analysis Chromosome 10 reference Chromosome 3 reference breakpoint breakpoint G-banding, mFISH a mBAND: der(10)t(3;10)(p21;q23) mBAND 10 mBAND 3 mFISH 10 3 10q23 3p21 Our strategy – K562 cell line Chromosome microdissection Real-time PCR assay Acute Leukemia Patients Patient 1 46,XX,t(X;4;11)(q25;q21;q23)[20] Fusion transcript MLL-AF4 =