THE PROSPECTIVE ANALYSIS OF LOW LEVEL BCR-ABL1 T315I MUTATION IN THE CD34+ CELLS OF DE NOVO CML PATIENTS

Konference: 2013 18th Congress of the European Hematology Association - účast ČR

Kategorie: Maligní lymfomy a leukémie

Téma: Chronic myeloid leukemia - Biology

Číslo abstraktu: P701

Autoři: Mgr. Tomáš Jurček; Ing. Filip Rázga, Ph.D.; Bc. Petra Mazancová; Ing. Dana Dvořáková, CSc.; Mgr. Marek Borský; MUDr. Daniela Žáčková, Ph.D.; Ing. Milena Musilová, Ph.D.; MUDr. Lukáš Semerád; prof. MUDr. Jiří Mayer, CSc.; Prof. MUDr. Zdeněk Ráčil, Ph.D.

Background:

The BCR-ABL1kinase domain (KD) mutations are associated with resistance to tyrosin kinase inhibitors (TKIs) and thereby with poor prognosis in chronic myeloid leukemia (CML) patients.Thus, an early detection of these mutations is important and could potentially leads to early therapeutic intervention and optimization of ongoing treatment regimen. The particular focus should be given to T315I mutation, which is resistant to all approved TKIs (imatinib, nilotinib and/or dasatinib). Considering the hierarchy of hematopoiesis, it should be expected that BCR-ABL1 KD mutations expand directly from stem cells or early progenitor cells. It has already been reported that these mutations were detected in these cells before their occurrence in bone marrow or peripheral blood.

Aims:

The prospective detection of low level T315I mutation in newly diagnosed CML patients, especially in early progenitor CML cells (CD34+; CD34-).

Methods:

The cell sorting (using a FACSVantage SE) of bone marrow CD34+ and CD34-cells from 50 de novo CML patients wasperformed at the time of diagnosis. Isolated RNA from sorted cells was reversibly transcribed into cDNA, which was subsequently used for amplification of BCR-ABL1 fusion gene followed by sensitive detection of T315I mutation by quantitative Ligation-PCR (ligPCRT315I). The estimated sensitivity of ligPCRT315I was 0.5% BCR-ABL1T315I/BCRABL1total.

Results:

Within our cohort of 50 de novo CML patients, low level T315I mutation was detected in 7/50 (14%) cases with obtained positivity closed to detection limit of applied method (~0.5%). These suspected samples were tested repeatedly to avoid any false-positive results. Moreover, additional CD34+/CD34- samples were tested for the presence of T315I mutation after the 3 months of TKI therapy. From these re-tested patients only 1/7 wasconsidered as positive for the low level T315I mutation. ThisT315I positive CML patient wassubsequently followed-up using ligPCRT315I analysis for the possible T315I mutation evolution. The expansion of T315I mutated clone was observed at the months 3 and 6 of TKI therapy with level of 0.7% and 4.2%, respectively. Other samples for T315I kinetics monitoring were not available since this patient died very quickly due to the progression of CML (8 months after diagnosis).

Summary and Conclusions:

Our prospective analysis of low level T315I mutation in early progenitor CML cells (CD34+; CD34-) of de novo CML patients showed that this key mutation does not frequently occur in CML patients at the time of diagnosis. We suppose that the presence and evolution of T315I mutation is more likely associated with increased genomic instability, advanced phase of CML or selective pressure of applied TKIs.

Supported by The Czech Leukemia Study Group for Life (CELL), the project (Ministry of Health, Czech Republic) for conceptual development of research organization 65269705 (University Hospital Brno, Brno, Czech Republic) and research grants (Ministry of Education, Youth and Sports, Czech Republic) MSMT0021622430 and MUNI/A/0723/2012

Abstrakta v časopise Haematologica 2013, Suppl1

Online Program

Datum přednesení příspěvku: 15. 6. 2013