Contributions
Abstract: EP659
Type: E-Poster Presentation
Session title: Chronic myeloid leukemia - Biology & Translational Research
Background
Chronic Myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the reciprocal translocation t(9;22)(q34;q21) resulting in the BCR-ABL1 fusion gene and the oncogenic protein P210 with constitutive tyrosine kinase activity. The study of this protein led to the development of tyrosine kinase inhibitors (TKI). It has been shown that patients with a deep and sustained molecular response could stop treatment. It is likely that relapse after TKI discontinuation is due to the persistence of Leukemic Stem Cell (LSC), which are transcriptionally quiescent. The expression of the enzyme dipeptidylpeptidase IV (CD26) is restricted to CD45+/CD34+/CD38- LSC in CML BCR/ABL1+, but is not found in other myeloid or lymphoid LSC. For this reason it is considered a novel specific biomarker of neoplastic cell in CML.
Aims
To detect the LSC (CD26+) in CML patients with different molecular responses (MR) and to assess if these cells remain in deep molecular response (DMR).
Methods
Peripheral blood samples from 174 CML patients (94 males and 80 females) were evaluated for detection of LSC by flow cytometry and quantitative PCR (qRT-PCR) for BCR-ABL1 rearrangement. All individuals provided an informed consent, in accordance with our Institutional Ethics Committee. The analysis of both studies was carried out simultaneously on the same sample, during the follow up at different time points after diagnosis and under TKI treatment (Imatinib, Nilotinib, Dasatinib). Median age was 48 years old (range 18-82). The molecular responses in International Scale (IS) were: 6% (10/174) Null MR, 9% (15/174) Minimal MR, 16% (28/174) Minor MR, 27% (47/174) Major MR and 42% (74/174) DMR (BCR-ABL1 transcripts reduction ≥ 4 log, including MR4.0, RM4.5 and RM5.0).
Flow cytometry studies were performed with 200 µl of whole blood stained with a panel of 8 antibodies: HLA-DR/CD45/CD38/CD26/CD34/CD117/CD123/CD3 using BD FACS Canto™ II flow cytometer. BCR-ABL1 qRT-PCR was performed by Taqman method with the Molecular MD One-Step kit using a Rotor-Gene 6000 Q real-time thermal-cycler (QIAGEN).
Results
The immunophenotype and molecular response were analyzed in all 174 cases. It was observed that patients with good molecular response (≥3log BCR-ABL1 reduction) had a significantly lower percentage of cases with LSC compared to those who had <3log reduction (P<0.0074, RO: 2.7). The analysis of the 74 patients with DMR (40% (29/74) in RM4.0, 55% (40/74) in RM4.5 and 5% (4/74) in RM5.0) showed that the LSC percentage was: 27% (8/29), 15% (6/41) and 0% (0/4) respectively. This suggests that the decrease in LSC could be related to deeper molecular responses.
Conclusion
Our results showed that CML patients with molecular response ≥3log BCR-ABL1 reduction were significantly associated with lower proportion of cases with persistence of LSC. When the LSC analysis was performed exclusively in cases with DMR, no significant differences were observed between RM4.0, RM4.5 and RM5.0. However, we observed that the presence of LSC decreased as the depth of the molecular response increased.
Since the LSC is highly quiescent, it could be present even in cases with undetectable DMR. In our study LSC were not detected in 81% (60/74) of cases in DMR, while in the remaining 19% (14/74), CD26+ cells remain despite DMR. This new approach to the study of the LSC could be useful during the follow up of patients in DMR, especially if they would be included in clinical trials of treatment discontinuation.
Keyword(s): Chronic myeloid leukemia, Leukemic stem cell, Molecular response
Abstract: EP659
Type: E-Poster Presentation
Session title: Chronic myeloid leukemia - Biology & Translational Research
Background
Chronic Myeloid leukemia (CML) is a myeloproliferative neoplasm characterized by the reciprocal translocation t(9;22)(q34;q21) resulting in the BCR-ABL1 fusion gene and the oncogenic protein P210 with constitutive tyrosine kinase activity. The study of this protein led to the development of tyrosine kinase inhibitors (TKI). It has been shown that patients with a deep and sustained molecular response could stop treatment. It is likely that relapse after TKI discontinuation is due to the persistence of Leukemic Stem Cell (LSC), which are transcriptionally quiescent. The expression of the enzyme dipeptidylpeptidase IV (CD26) is restricted to CD45+/CD34+/CD38- LSC in CML BCR/ABL1+, but is not found in other myeloid or lymphoid LSC. For this reason it is considered a novel specific biomarker of neoplastic cell in CML.
Aims
To detect the LSC (CD26+) in CML patients with different molecular responses (MR) and to assess if these cells remain in deep molecular response (DMR).
Methods
Peripheral blood samples from 174 CML patients (94 males and 80 females) were evaluated for detection of LSC by flow cytometry and quantitative PCR (qRT-PCR) for BCR-ABL1 rearrangement. All individuals provided an informed consent, in accordance with our Institutional Ethics Committee. The analysis of both studies was carried out simultaneously on the same sample, during the follow up at different time points after diagnosis and under TKI treatment (Imatinib, Nilotinib, Dasatinib). Median age was 48 years old (range 18-82). The molecular responses in International Scale (IS) were: 6% (10/174) Null MR, 9% (15/174) Minimal MR, 16% (28/174) Minor MR, 27% (47/174) Major MR and 42% (74/174) DMR (BCR-ABL1 transcripts reduction ≥ 4 log, including MR4.0, RM4.5 and RM5.0).
Flow cytometry studies were performed with 200 µl of whole blood stained with a panel of 8 antibodies: HLA-DR/CD45/CD38/CD26/CD34/CD117/CD123/CD3 using BD FACS Canto™ II flow cytometer. BCR-ABL1 qRT-PCR was performed by Taqman method with the Molecular MD One-Step kit using a Rotor-Gene 6000 Q real-time thermal-cycler (QIAGEN).
Results
The immunophenotype and molecular response were analyzed in all 174 cases. It was observed that patients with good molecular response (≥3log BCR-ABL1 reduction) had a significantly lower percentage of cases with LSC compared to those who had <3log reduction (P<0.0074, RO: 2.7). The analysis of the 74 patients with DMR (40% (29/74) in RM4.0, 55% (40/74) in RM4.5 and 5% (4/74) in RM5.0) showed that the LSC percentage was: 27% (8/29), 15% (6/41) and 0% (0/4) respectively. This suggests that the decrease in LSC could be related to deeper molecular responses.
Conclusion
Our results showed that CML patients with molecular response ≥3log BCR-ABL1 reduction were significantly associated with lower proportion of cases with persistence of LSC. When the LSC analysis was performed exclusively in cases with DMR, no significant differences were observed between RM4.0, RM4.5 and RM5.0. However, we observed that the presence of LSC decreased as the depth of the molecular response increased.
Since the LSC is highly quiescent, it could be present even in cases with undetectable DMR. In our study LSC were not detected in 81% (60/74) of cases in DMR, while in the remaining 19% (14/74), CD26+ cells remain despite DMR. This new approach to the study of the LSC could be useful during the follow up of patients in DMR, especially if they would be included in clinical trials of treatment discontinuation.
Keyword(s): Chronic myeloid leukemia, Leukemic stem cell, Molecular response