Sensitivity of AML Cells with FLT3to PKC412 In Vitro / 中国实验血液学杂志

<p><b>OBJECTIVE</b>To explore the clinical value of PKC412 (midostaurin) in treatment of AML patients with FLT3.</p><p><b>METHODS</b>The bone marrow or peripheral blood were collected and heparinized from 21 newly diagnosed FLT3AML patients, then the mononuclear cells from bone marrow or peripheral blood were isolated by density-gradient method. The sensitivity of leukemia cells to PKC412 of 8 concentration in vitro was detected by ATP-bioluminescence-tumor chemosensitivity assay (ATP-TCA), and the relationship among sensitivity results in vitro, risk stratification and therapeutic efficacy was analyzed.</p><p><b>RESULTS</b>The leukemia cells of 21 patients with AML displayed different sensitivities to PKC412 in vitro. The rate of sensitivity in vitro was 42.9%, and sensitive concentration in vitro were between 1 µmol/L and 5 µmol/L. There was no significant relationship between risk stratification and sensitivity results of PKC412 in vitro. There was also no significant relationship between clinical efficacy and sensitivity results of PKC412 in vitro. The survival of patients in low-risk and intermediate-risk groups was better than that of patients in high-risk groups (P=0.015).</p><p><b>CONCLUSION</b>PKC412 can be one of the effective therapeutic method for AML patients without FLT3 mutation. The sensitivity of leukemia cells to PKC412 may become a prognostic marker for evaluating clinical efficacy of PKC412, which is independent of other factors.</p>

Genetic characteristics of human acute lymphoblastic leukemia cell line Molt-4 / 中国实验血液学杂志

This study was aimed to investigate the genetic characteristics of human acute lymphoblastic leukemia cell line Molt-4, and evaluate its application in measuring telomere length by Flow-FISH. Molt-4 cell line was cultured in suspension and subcultured regularly. Eight different passages of Molt-4 cells in exponential stage were selected.The growth curves were drawn by cell counting method, meanwhile calculating the population doubling times of cells,DNA ploidies were determined by flow cytometry,karyotypes were analyzed by G-banding and telomere lengths were measured by Southern blot. The results showed that the population doubling time of Molt-4 cell line was (1.315 ± 0.062) d, DNA ploidy index was (2.085 ± 0.0093) , and the telomere length was (32.05 ± 5.27) kb. There were no significant difference among different passages (P = 0.931,0.888 and 0.935 separately). The karyotypes showed that the chromosome numbers of Molt-4 cell line were from 91 to 99 in different metaphases, and the majority of them were hypertetraploid, and stable and recurrent structural abnormalities of chromosomes could be kept. It is concluded that the stable genetic characteristics and the longer telomere length of Molt-4 cell line makes it be a feasible control cells in measurement of telomere length by Flow-FISH.

Application of flow cytometry-fluorescence in situ hybridization in the measurement of relative telomere length of bone marrow CD34(+) cells in patients with myelodysplastic syndrome / 中国实验血液学杂志

This study was aimed to investigate the feasibility of flow cytometry-fluorescence in situ hybridization (Flow-FISH) in measuring the telomere length of bone marrow cell subgroups in patients with myelodysplastic syndrome (MDS). Seven newly diagnosed patients with low-risk MDS and seven nutritional anemia patients who were matched with age and sex, were enrolled in this study. Heparinized bone marrow were sampled. Taking Molt-4 cell line as internal control cells, leukocytes isolated from whole bone marrow were labeled with CD34-Alexa Fluork ® 647, then denatured by high temperature and hybridized with FITC-conjugated telomere probe. The DNA was counterstained and the relative telomere length (RTL) of nucleated cells and CD34(+) cells in bone marrow were measured by four-color flow cytometry. The results showed that CD34(+) cells could be gated for the measurement of RTL in both groups, undergoing the denaturation and hybridization. Primary analysis indicated that the RTL of bone marrow CD34(+) cells in MDS patients was significantly shorter than that of bone marrow nucleated cells (P = 0.001), and the RTL of both CD34(+) cells and nucleated cells in bone marrow of MDS patients were significantly shorter than that of control group (P = 0.020, 0.002). It is concluded that the application of Flow-FISH in the measurement of RTL of certain cell subgroup is feasible by labeling the cell with thermostable fluorescence-conjugated antibody, and this technique is worthy to be investigated further.

Application of interphase FISH on cell smears in detection of hematological diseases / 中国实验血液学杂志

The study was aimed to investigate the application value of interphase fluorescence in situ hybridization (FISH) on cell smears in hematological diseases. Both interphase FISH on peripheral blood smears and bone marrow smears treated by methanol/acetic acid, and routine interphase FISH of bone marrow cells dropped on slides were done at the same time, in order to detect Ph chromosome by BCR/ABL dual color, dual fusion probe in 20 patients with chronic myelogenous leukemia or acute lymphoblastic leukemia which had been proven to display Ph chromosome positive. The results indicated that as compared with routine interphase FISH, the interphase FISH on cell smears could also offer reliable result. It is concluded that interphase FISH on cell smears is a kind of reliable and time-saving technique, which is also suitable for retrospective research and worthy to further apply in clinic.

Multiparameter flow cytometric evaluation of bone marrow involvement in B cell non-Hodgkin's lymphoma / 中国实验血液学杂志

The objective of study was to evaluate the clinical values of multiparameter flow cytometry (MPFC) and cytomorphology of bone marrow aspiration(BMA) in detecting bone marrow involvement in patients with B cell Non-Hodgkin's lymphoma (B-NHL). 96 bone marrow samples from the patients with B-NHL were measured by MPFC using CD45/SSC and CD20/SSC gating strategy combined with anti-kappa and anti-lamda monoclonal antibodies, and then compared with results acquired by cytomorphologic analysis of BMA. The results showed that the bone marrow involvement was confirmed by MPFC in 38 cases (39.6%), while it was detected by cytomorphologic analysis of BMA only in 12 cases (12.5%). There was a significant difference between the two methods (p<0.05). 12 positive cases detected by cytomorphologic analysis of BMA were also positive by MPFC. There was no difference of 3-year overall survival rate between negative and positive cases detected by MPFC, but their 4-year overall survival rate was 73.18+/-6.65% and 44.13%+/-19.55% respectively (p<0.05). It is concluded that the MPFC is a more sensitive method for detecting bone marrow involvement in patients with B-NHL than cytomorphologic analysis of BMA. The 4-year overall survival rate of the patients without bone marrow involvement was significant higher than those of patients with bone marrow involvement. Bone marrow involvement in B-NHL detected by MPFC can be useful for clinical evaluation and prognosis prediction.

Prognosticating relapse risk based on multiparameter flow cytometric assessment of minimal residual disease in patients with acute myeloid leukemia / 中国实验血液学杂志

The objective of this study was to investigate the prognosticating value of multiparameter flow cytometry in detection of minimal residual disease (MRD) and relapse risk of patients with acute myeloid leukemia (AML). Multiparameter flow cytometry (MPFC) analysis was used to detect the leukemia-associated aberrant immunophenotype (LAIP) of the pretreated patients with AML and to assess the levels of MRD after remission induction (Post-Ind MRD) and consolidation therapy (Post-Cons MRD). The results showed that the definite LAIP could be detected in 94.3% of the patients (115/122) with AML (except APL). Among 115 cases only one LAIP was identified in 15 cases (13.0%), but two or more LAIP were identified in other 100 cases (87.0%). The most frequent LAIP identified was cross-lineage antigen expression (40.9%). The percentages of asynchronous antigen expression, antigen over-expression and antigen lack expression were 20.9%, 27.0%and 34.8% respectively. MRD frequency was monitored in 41 AML patients with CR after remission induction chemotherapy and 2 or more cycles of consolidation chemotherapy. 24 patients were Post-Ind MRD(+) and 17 patients were Post-Ind MRD(-). The percentages of relapse in cases of Post-Ind MRD(+) and Post-Ind MRD(-) were 75.0% (18/24) and 29.4% (5/17) respectively after consolidation chemotherapy. The relapse free survival (RFS) times of the patients with Post-Ind MRD(+) and Post-Ind MRD(-) were 49.06 +/- 6.53 months and 11.92 +/- 1.64 months (p < 0.0001) respectively. 18 patients were Post-Cons MRD(+) and 23 patients were Post-Cons MRD(-). The percentages of relapse in cases of Post-Cons MRD(+) and Post-Cons MRD(-) patients were 100% (18/18) and 21.7% (5/23) respectively after consolidation chemotherapy. The RFS times of the patients with Post-Cons MRD(+) and Post-Cons MRD(-) were 41.74 +/- 5.52 months and 10.06 +/- 1.72 months (p < 0.0001) respectively. It is concluded that the levels of post-Ind MRD and post-Cons MRD identified in the patients with AML was highly associated with their RFS. The detection of MRD by MPFC provides prognostic information in AML patients.

Aberrant immunophenotypes in 126 patients with acute leukemia / 中国实验血液学杂志

The existence of leukemia aberrant immunophenotypes (LAIP) has been suggested to be a valuable tool for the detection of minimal residual disease (MRD), as they could distinguish leukemic cells from normal hematopoietic progenitors. This study was purposed to analyze the characteristics of LAIP in acute leukemia and further explore the proportion of different types of LAIP in acute leukemia patients. Flow cytometry (FCM) with four color and CD45/SSC gating were used to detect the antigen expression in samples of bone marrow from 126 patients with acute leukemia. The results showed that definite LAIP could be detected in about 76% patients. The LAIP could be divided into four groups as cross-lineage antigen expression, asynchronous antigen expression, antigen overexpression and antigen lack expression. The percentages of these LAIPs were 39%, 46%, 21% and 29% respectively. About 11% out of analyzed cases showed the existence of only one aberrant phenotype while two or more of aberrant phenotypes could be detected in majority cases. It is concluded that the LAIP with four subgroups can be detected in the majority of patients with acute leukemia and immunophenotyping based on LAIP is applicable for the detection of MRD.