Dihydroartemisinin treatment of multiple myeloma cells causes activation of c-Jun leading to cell apoptosis.

The aim of the present study was to investigate the effect of dihydroartemisinin (DHA) on a multiple myeloma cell line. An MTT assay, flow cytometry and reverse transcription-polymerase chain reaction (RT-PCR) were used for the analysis of cell viability, cell cycle distribution and c-Jun N-terminal kinase (JNK) expression, respectively. Treatment of U266 cells using DHA caused a significant (P<0.05) decrease in cell viability compared with the control cells. An increase in the concentration of DHA from 1 to 100 µmol/l reduced cell viability from 87 to 35% compared with 100% in the control cultures at 48 h. A significant (P<0.05) increase was observed in the sub-G0/G1 phase population of the U266 cells with an increase in DHA concentration from 1 to 100 µmol/l. Treatment with 1, 3, 10, 30 and 100 µmol/l concentrations of DHA increased the sub-G0/G1 phase cell population to 3.13, 8.25, 24.91, 31.47 and 38.54%, respectively. RT-PCR analysis of DHA-treated or -untreated U266 cells after 48 h demonstrated a significant (P<0.01) increase in caspase-3 expression. Treatment of the cells for 48 h with DHA led to a significant increase in c-Jun expression. DHA treatment at 1, 3, 10, 30 and 100 µmol/l concentrations caused an increase in the level of c-Jun by 0.174±0.001, 0.254±0.002, 0.387±0.001, 0.502±0.003 and 0.679±0.005, respectively, compared with 0.982±0.001 in the control cells. The addition of SP600125 to the cells incubated with DHA resulted in a significant decrease in the caspase-3 and c-Jun expression levels compared with those cells incubated with DHA alone. These findings confirm that treatment with DHA increased caspase-3 and c-Jun expression in the U266 cells through activation of the JNK signaling pathway. Thus, DHA inhibited proliferation of multiple myeloma cells by interfering with the JNK signaling pathway.

Deep molecular responses achieved in chronic myeloid leukemia in chronic phase patients with BCR-ABL1 >10% at 3 months who are early switched to nilotinib.

OBJECTIVES: There are still controversies about whether or when to change therapy for those chronic myeloid leukemia in chronic phase (CML-CP) patients with BCR-ABL1 transcript level at 3 months higher than 10%. Here we studied the clinical significance of early switching to nilotinib in CML-CP patients with BCR-ABL1 >10% at 3 months. METHODS: We investigated 495 first-line imatinib-treated patients with CML-CP and identified 117 (23.6%) patients with BCR-ABL1 >10% at 3 months, including 46 patients with warning response defined according to ELN-2013 recommendations. In 46 patients with warning response, 26 of them continued imatinib therapy and 20 patients early switched to nilotinib therapy. RESULTS: Compared to continued imatinib group, nilotinib group showed significantly higher percentage achieving BCR-ABL1 <1% at 6 months (85.0% vs. 19.2%, P = 0.0004), better cumulative rates of MR3.0 and MR4.0 of 4 years (82.1% vs. 41.2%, P = 0.0091; 61.5% vs. 18.6%, P = 0.035). CONCLUSION: In summary, early switching to nilotinib enabled more CML-CP patients with warning molecular response at 3 months to achieve early and deeper molecular response vs. remaining on imatinib.

Chronic myeloid leukemia with e14a3 BCR-ABL transcript: analysis of characteristics and prognostic significance.

Chronic myeloid leukemia (CML) is characterized by Philadelphia chromosome (Ph) and BCR-ABL fusion genes. This study retrospectively analyzed 2381 CML patients with Ph chromosome confirmed by cytogenetics, Fluorescence in situ hybridization (FISH) or real-time quantitative polymerase chain reaction (Q-PCR). Among them, five CML patients without e13a2, e14a2 or e1a2 transcripts detected by Q-PCR were identified. DNA sequencing confirmed the fusion of BCR exon 14 and ABL exon 3. Case 1 reponded poorly to imatinib and achieved complete cytogenetic response (CCyR) after converting from imatinib to dasatinib. BCR-ABL transcripts were undetectable in cases after 2, 3 and 4 treated with imatinib after 6, 6 and 3 months, respectively, and in one patient who had undergone allogeneic hematopoietic stem cell transplantation after 4 months. Q-PCR may miss the detection of rare cases that are not covered by the primers used in Q-PCR, unless the proper primers are used.

[A clinical and laboratory study of chronic myeloid leukemia with atypical BCR-ABL fusion gene subtypes].

OBJECTIVE: To explore the clinical and laboratory features of chronic myeloid leukemia (CML) with atypical e14a3 and e19a2 BCR-ABL fusion gene subtypes. METHODS: We retrospectively analyzed a cohort of CML patients with Ph chromosome positive confirmed by cytogenetic and FISH but classical e13a3(b2a2), e14a2(b3a2)and e1a2 fusion transcripts negative identified by conventional real-time quantification RT-PCR (RQ-PCR). Further RQ-PCR was done with the forward primer and reverse primer designed to detect rare atypical BCR-ABL fusion genes including e14a3 and e19a2 transcripts. Direct sequencing analysis was performed on the PCR products and mutations in the BCR-ABL kinase domain were detected. The clinical data of patients were retrospectively analyzed. RESULTS: Six CML patients were found to carry t(9;22) abnormality and BCR-ABL rearrangement confirmed by FISH but classical BCR-ABL fusion genes negative detected by RQ-PCR. Further RQ-PCR and sequencing analysis confirmed the fusion of BCR exon 14 and ABL exon 3 in five CML patients (case 1-5) and the fusion of BCR exon 19 and ABL exon 2 in one CML patient (case 6). E255K and I293T IM-resistant mutations were detected in case 1 and 2, respectively. Among five cases with e14a3 transcripts, four were CML-CP, one CML-AP. Four patients were male and one was female. The median age was 48 years. The patient (case 6) with e19a2 transcripts was 40-year-old female with a diagnosis of CML-CP and PLT count was more than 1 000×109/L. Imatinib (IM) therapy was administer in case 1, 2, 3, 4 and hematopoietic stem cell transplantation (HSCT) was undergone in case 5 after hydroxyurea (Hu) or interferon failure. Case 1 who had E255K IM resistant mutation, responded poorly to IM but obtained a complete cytogenetic remission (CCyR) after a substitution of dasatinib for IM. Case 2 and 3 achieved CCyR 6 months later after IM treatment and had been maintained well with IM despite I293T mutation in case 2. Case 4 attained CCyR 3 months later after IM treatment but relapsed and died soon. Case 5 was still in CCyR after HSCT. Case 6 with e19a2 transcripts got complete hematologic response after Hu treatment and CCyR was achieved soon after IM therapy. CONCLUSION: Incidence of CML with atypical transcripts is extremely low. They could benefit from tyrosine kinase inhibitors or HSCT. Rare and atypical BCR- ABL fusion gene subtypes could be missed by conventional RQ-PCR.