[Detection and Diagnostic Values of JAK2, CALR, MPL Gene Mutations in 208 Cases of BCR/ABL1 Negative Chronic Myeloproliferative Diseases].

OBJECTIVE: To detect the JAK2, CALR and MPL gene mutations in patients with BCR/ABL1 negative chronic myeloproliferative diseases（BCR/ABL1-CMPD）and to evaluate their diagnostic value. METHODS: Two hundred and eight cases of BCR/ABL1-CMPD comprising of 146 cases of essential thrombocythemia（ET）, 37 cases of polycythemia vera（PV）and 25 cases of primary myelofibrosis（PMF）from March 2012 to December 2015 were enrolled in the BCR/ABL1-CMPD, while 124 cases of secondary thrombocythemia and 73 cases of secondary polycythemia were enrolled in the control group. The genomic DNA and total RNA Were isolated from bone marrow or peripheral blood, then the exons 12 to 20 of JAK2 gene, exon 10 of MPL gene and exons 3 to 9 of CALR gene were analyzed by using DNA sequencing. RESULTS: among 146 ET patients, the JAK2, CALR or MPL mutations were found in: 138 cases（94.5%）including 86 cases with JAK2V617F mutation（58.9%）and 2 cases（1.4%）with exon 12 of JAK2 mutations. CALR mutations were detected in 41 cases（28.1%）, among them type 1（c.1092_1143del）in 22 cases, type 2（c.1154_1155insTTGTC）in 11 cases, and type 5（c. 1091_1142del）, type 8（c.1104_1137del）, type 41(c.1107_1137del）, type 42(c.1125_1125del）in one case respectively. In addition, 4 cases were detected withother mutations of the CALR gene（c.1107_1115del, c.1111_1144 del, c.1101 A>C, c.1112_1117del）. Moreover, 9 cases harbored MPL mutations（6.2%）. Secondly, 31 patients were detected with JAK2V617F mutation（83.8%）in 37 cases of PV, and JAK2 exon 12 mutations were found in 2 cases（5.4%）. Besides, CALR mutations were detected in 2 cases（5.4%）, including 1 case of type I, the other of novel mutation of CALR. Thirdly, 19 in 25 cases of PMF were detected with JAK2V617F mutation（76%）, 2 cases with CALR mutations（8%）. 4 patients（16%）, JAK2, CALR or MPL mutations were not detected, but among them 3 cases were found harboring other genetic abnormalities. Fourthly, no mutations of JAK2, MPL and CALR genes were detected in 124 patients with secondary thrombocytosis and 73 cases with secondary polycythemia. CONCLUSION: Combined detection of JAK2, CALR and MPL gene mutations can cover the vast majority of patients with BCR/ABL1-negative myeloproliferative neoplasms. For higher frequencies of the mutations of CALR in ET patients, CALR mutation can be used as a new diagnostic marker in ET patients with JAK2 and MPL wild type.

Detection of intracellular IgD using flow cytometry could be a novel and supplementary method to diagnose IgD multiple myeloma.

BACKGROUND: We examined whether detecting the heavy chain of cytoplasmic immunoglobulin D (IgD) by flow cytometry could be used as a supplemental method to diagnose IgD multiple myeloma (MM). METHODS: Bone marrow (BM) samples of thirty-five patients with MM were collected. Five of them were IgD MM, the rest of thirty were other subtypes of MM. Antibodies to four types of heavy chains of immunoglobulin (e.g., IgA, IgG, IgM, and IgD) were analyzed by flow cytometry in each patient's BM sample. RESULTS: The five IgD MM patients were all positive for cytoplasmic IgD. The percentage of IgD positive MM cells among nucleated cells varied from 0.4 to 12.9%. Cytoplasmic IgG was positive in eight patients with IgG MM (n = 9); cytoplasmic IgA was positive in all patients with IgA MM (n = 10); cytoplasmic IgM was positive in one patient with IgM MM (n = 1). No heavy chain was detected in light chain MM (n = 9) and non-secretory subtype (n = 1). CONCLUSIONS: Detection of cytoplasmic IgD by flow cytometry is a convenient, sensitive and supplemental method to diagnose IgD MM.

Non-malignant T-cells lacking multiple pan-T markers can be found in lymph nodes.

In order to observe and ascertain the properties of a sub-group of T cells in the lymph node (LN) from seven patients who did not suffer from T cell lymphoproliferative disorders (T-LPDs), the expression levels of several pan-T markers were evaluated by multiparameter flow cytometry (FC) and the clonality of these T-cells was evaluated by both FC analysis and PCR assessment. It turned out that multiple pan-T-cell markers such as CD2, CD5 and CD7 were found to be lost in these T cells. The majority of them were positive for TCRαß, only a minority of them being positive for TCRγδ. A subset of these T-cells were positive for CD4 or CD8 or dual-negative for CD4 and CD8. Oligoclonality was detected in one case by FC, while clonal TCR rearrangement was detected in three cases. Absence of multiple pan-T-cell markers could be found in benign T cells in LNs.

[Clinical Features and Prognosis of t(8;21) AML Patients in China: A Multicenter Retrospective Study].

OBJECTIVE: To summarize the clinical characteristics of peripheral blood, immune phenotypes, fusion genes and cytogenetics of patients with t(8;21) acute myeloid leukemia(AML) through the retrospective analysis of 586 patients with t(8;21) AML from 15 blood disease research centers in Northern area of China. METHODS: The factors affecting prognosis of patients with t(8;21) AML were investigated by using univariate and multivariate COX regression. RESULTS: The immune type of t(8;21) AML patients was mainly with HLA-DR+, CD117+, CD34+, MPO+, CD38+, CD13+ and CD33+ (>95%), part of them with CD19+ and CD56+; the most common accompanied mutation of t(8;21) AML patients was C-KIT mutation (37.8%); in addition to t(8;21) ectopic, the most common chromosomal abnormality was sex chromosome deletions (38.9%). The univariate analysis revealed a significant survival superiority of OS and PFS in t(8;21) AML patients of WBC≤3.5×109/L without C-KIT mutation, the newly diagnosed ones achieved HSCT(P<0.05), only survival superiority on OS in t(8;21) AML patients with extramedullary infiltration and CD19 positive; the results of multivariate analysis showed a significant survival superiority on OS and PFS in t(8;21) AML patients with WBC≤3.5×109/L(P<0.05). CONCLUSION: The clinical features of t(8;21) AML patients in China are similar to those in other countries, WBC≤3.5×109/L is a good prognostic factor while the C-KIT mutation is a poor one in t(8;21) AML patients.

Comparison of Clinical Efficacy of Cytarabine with Different Regimens in Postremission Consolidation Therapy for Adult t(8;21) AML Patients: A Multicenter Retrospective Study in China.

BACKGROUND: The survival of patients with acute myeloid leukemia (AML) with t(8;21) was reported to be shorter in China than in other countries. PATIENTS: We analyzed the correlation between different cytarabine (Ara-c) regimens and outcome in 255 t(8;21) AML patients in China who received postremission consolidation chemotherapy only. RESULTS: The 5-year overall survival (OS) of the high-dose Ara-c group (HDAC; 2≤ Ara-c ≤3 g/m2), intermediate-dose Ara-c group (MDAC; 1.0≤ Ara-c <2.0 g/m2), low-dose Ara-c group (LDAC; 0.2< Ara-c <1.0 g/m2) and standard-dose Ara-c group (SDAC; 0.1≤ Ara-c ≤0.2 g/m2) were 65.3, 39.4, 25.2 and 27.9%, respectively (p = 0.003). In the HDAC group, but not in the MDAC group, the 5-year OS of patients who achieved 3-4 cycles of chemotherapy was superior to those who underwent 1-2 cycles (84.4 vs. 43.6%, p < 0.05), and the 3-year OS of patients who achieved an accumulated 36 g/m2 of Ara-c was significantly higher compared to those who did not (85.3 vs. 39.2%, p < 0.05). Multivariate analysis indicated that factors such as WBC >3.5 × 109/l, PLT ≤30 × 109/l, and extramedullary infiltration were associated with a poor prognosis. CONCLUSION: The survival of t(8;21) AML patients treated with high-dose Ara-c (≥2 g/m2) was superior to other dose levels in postremission consolidation chemotherapy. Patient survival was improved by 3-4 cycles of chemotherapy with an accumulated concentration of 36 g/m2 of Ara-c. WBC >3.5 × 109/l, PLT ≤30 × 109/l and extramedullary infiltration could be indicative of a poor clinical prognosis.

[Application of Cytogenetic Test for Diagnosis of Bone Marrow Involvement in Patients with Non-Hodgkin's Lymphoma].

OBJECTIVE: To investigate the role of cytogenetic analysis in the detection of bone marrow (BM) involvement in patients with non-Hodgkin's lymphoma (NHL). METHODS: The bone marrow samples of 74 patients with NHL were detection by using morphology, cytogenetic test, flow cytometry and molecular biological assay. The detected results of morphology, cytogenetic test, flow cytometry and molecular biological assay alone and thier combined detection were compared, the detective rate and consistencies of the 4 methods were analyzed. RESULTS: The detection rates of BM involvement by using morphology, cytogenetic, flow cytometry, and molecular biological assays were 21.6%, 17.6%, 23.0% and 33.8% respectively. The detective rate was enhanced to 44.6% by combining the 4 methods. Cytogenetic test showed the result consistent with the other methods. CONCLUSION: Although cytogenetic test shows a lower detective rate than the other methods, but in some patients the cytogenetic test can detect the abnormality of bone marrow which can not be detected by other methods alone, the combination test of 4 detection methods can enhance the detectable rate of BM involvement.

The application of CD73 in minimal residual disease monitoring using flow cytometry in B-cell acute lymphoblastic leukemia.

The expression of CD73 by flow cytometry (FC) in bone marrow (BM) specimens of B-cell acute lymphoblastic leukemia (B-ALL) with or without minimal residual disease (MRD) was studied, and its advantages were evaluated using the MRD assay. This study also detected the expression profile of CD73 in hematogones and mature B cells in BM specimens of 18 healthy donors. Results showed that the mean value of CD73 expression in MRD-positive B cells was 6-fold greater than that in the MRD negative ones. Also, 41.82% MRD-positive B-ALL cases expressed high CD73 and the sensitivity of CD73-based MRD detection reached 10(-4). Since the expression of CD73 increases with the maturation of normal B cells, it is better to mix it with CD34, CD10 and CD20 in one tube to prevent the disturbance of mature B cells. CD73 is recommended as an optional MRD marker for B-ALL patients by using FC.

[Significance of CD37 expression in malignant B cells].

The aim of this study was to clarify the clinical significance of CD37 expression in B cells from B acute lymphoblastic leukemia (B-ALL) and B cell non-Hodgkin's lymphoma (B-NHL). The expression level of CD37 on B cells from bone marrow samples of normal controls (n = 19), B-ALL patients [including untreated cases (n = 5) and cases with minimal residual disease (MRD, n = 15)] and B-NHL patients (n = 25) whose bone marrow was involved by lymphoma cells, was detected by multiple parameter flow cytometry. The results indicated that the B cells from both untreated cases and cases with MRD lowly expressed CD37 (1.04 ± 0.24 and 1.50 ± 0.89), the normal precursor B cells (control cases) also lowly expressed CD37 (1.64 ± 0.52). There was no difference of CD37 expression level between 3 groups of cases(P > 0.05). Meanwhile the normal mature B cells and B-NHL cells highly expressed CD37 (14.23 ± 7.84 and 14.53 ± 10.93), but there was no difference of CD37 expression between them (P > 0.05). The comparison of CD37 expression level in normal B cells of development stages showed that the progenitor B cells lowly expressed CD37 (0.88 ± 0.17), the CD37 expression of precursor B cells was enhanced (2.44 ± 0.69), while the CD37 expression level of mature B cells was highest. It is concluded that the low expression of CD37 is not the characteristic of B- ALL cells. The expression level of CD37 increases gradually during the mature process of B cells, i.e, the expression level of CD37 does not associate with benignity or malignancy of B cells.

[Mechanism of antitumor effect of ursolic acid on T (8;21) leukemia cell kasumi-1].

This study was purposed to investigate the anti-tumor effect of ursolic acid (UA) on t(8;21) leukemia cell line kasumi-1 and its possible mechanisms. The kasumi-1 cells were treated with UA at different concentration for different duration of time. The growth inhibition of kasumi-1 treated with UA was detected by using CCK-8 test, and the morphological changes of kasumi-1 cells were observed by Wright's staining. Furthermore, the apoptosis rate of kasumi-1 was examined by flow cytometry. Lastly, the expression of AML1-ETO, KIT, MYC, CCND1, BCL-2, P53, BAX, MDM2 and protein were detected by using real-time quantitative PCR and Western blot respectively. The results showed that the UA obviously inhibited the growth of kasumi-1 cells in dose- and time-dependent manners. The apoptotic morphological changes of cells were presented when kasumi-1 cells were treated with UA for 48 hours. The apoptotic rate of kasumi-1 cells increased in a dose- and time-dependent ways, and the mRNA levels of AML1-ETO, KIT, MYC, CCND1, BCL2, MDM2 decreased in kasumi-1 cells treated with UA, as well as the protein levels. Meanwhile, UA up-regulated the mRNA and protein levels of P53 in the same manner. It is concluded that UA can exert its anti-tumor effect by inhibiting the proliferation and inducing the apoptosis of kasumi-1 cells in a dose-and time-dependent manners, that may provide the clues for a new targeting therapy to t(8;21) leukemia.

[Mechanism concerning antitumor effect of oridonin on multiple myeloma cell line U266].

This study was purposed to investigate the antitumor effect of oridonin on human multiple myeloma cell line U266 and its possible mechanism. The CCK-8 test was used to determine the inhibitory effect of oridonin on proliferation of U266 cells. The morphological changes of U266 cells were observed under optical microscope. The apoptosis rate of U266 cells was detected by flow cytometry. The mRNA levels of FGFR3, BCL2, CCND1 and MYC genes were quantified by using real-time quantitative PCR method, and the protein levels of BCL2, MYC, CCND1, FGFR3 and P53 were detected by Western blot. The results showed that the oridonin obviously inhibited the growth of U266 cell in dose-and time-dependent manners. As for morphological changes, characteristic apoptotic cells presented in U266 cells treated with 10 µmol/L oridonin for 24 hours. The apoptotic rate of U266 cells increased in dose and time dependent manners; after treatment of U266 cells with oridonin the mRNA levels of FGFR3, BCL2, CCND1 and MYC as well as the their protein levels decreased. Occasionally, the oridonin up-regulated the protein levels of P53 in the same manner. It is concluded that the oridonin can exert its anti-tumor effect by inhibiting proliferation and inducing apoptosis of U266 cell in dose dependent and time dependent manners, that maybe give the clues about new program of target therapy for multiple myeloma.

[Significance of magnetic resonance imaging in the detection of iron overload].

OBJECTIVE: To evaluate the significance of magnetic resonance imaging (MRI) T2(*) value analysis in patients with iron overload and compare it with other clinical parameters. METHODS: A total of 53 patients with suspected iron overload were recruited from four Beijing hospitals from December 2010 to December 2012. Their liver and heart T2(*) values were calculated and their serum ferritin (SF), transferrin saturation, blood transfusion volume and other clinical parameters were recorded and analyzed. RESULTS: There were 37 males and 16 females with a medium age of 50 years(15-72 years). Their etiologies included myelodysplastic syndromes (MDS, n = 25), aplastic anemia (AA, n = 16), myelofibrosis (n = 5), hemochromatosis (n = 2) and ß thalassaemia (n = 2), and 3 patients with high SF values were found on regular health examinations. Among them, there were transfusion history (n = 45), SF>1000 µg/L (n = 49), sign of iron overload (n = 10), abnormal liver function (n = 38) and hyperglycemia (n = 32). T2(*) value analysis showed that 10 patients had no evidence of iron overload, 43 patients had liver iron overload (14 mild, 22 moderate and 7 severe) and 2 patients had heart iron overload (1 MDS with heavy transfusion history and 1 AA with heart failure). No relations existed between T2(*) value and SF (P = 0.050) , T2(*) value and transfusion volume (P = 0.820) , and liver T2(*) value and heart T2(*) value (P = 0.129) . CONCLUSIONS: MRI T2(*) value is an accurate way of quantitative detection of iron overload. It provides a comprehensive understanding of patients with iron overload in conjunctions with MRI T2(*) value and other clinical parameters.

[Analysis of cytogenetic characteristics in patients with myelodysplastic syndrome].

This study was aimed to investigate the distribution of chromosomal aberrational karyotype in myelodysplastic syndrome (MDS) subgroups, the characterizations of numerical and structural aberration. The chromosome was prepared with simple culture of bone marrow, and the karyotype was analysed by G banding technique. The results showed tht 54 out of 127 patients (42.5%) had clonal chromosome aberrations, and the abnormal rates were different in subgroups: 30% (3/10) in MDS-RA, 35.9% (23/64) in MDS-RCMD, 22.2% (2/9) in MDS-RAS, 45% (9/20) in MDS-RAEB-I, 66.7% (14/21) in MDS-RAEB-II, 100% (3/3) in 5q-syndrome, respectively. Among 54 abnormal chromosome patients, 21 patients showed numerical aberration, 14 patients showed structural aberration, and the other 19 patients showed both numerical and structural aberration. The order of frequent aberrations was as follows complex karyotype (11.02%, 14/127), single +8 (10.24%, 13/127), -7/7q- (3.9%, 5/127), 1q+ (3.15%, 4/127), -X/-Y (3.15%, 4/127), 20q- (2.36%, 3/127), 5q- (2.36%, 3/127). The frequency of complex karyotype in MDS-RAEB (including RAEB-I and RAEB-II) was higher than that in non MDS-RAEB (including RA, RCMD, RAS, 5q-syndrome) (P < 0.05), and the frequency of balanced translocation was lower than that in non-balanced translocation (P < 0.05), and both of the two balanced translocation patients were found in MDS-RAEB. It is concluded that MDS is highly heterogeneous clonal disorder, a great majority of cytogenetic changes can be detected and most of which are recurrent aberrations, balanced translocations are rare, and only found in MDS-RAEB. The frequency of complex karyotype in MDS-RAEB is higher, and the patients with dup (1) (q21q32) recurrent abnormality is common in this study.

[Effect of NKG2D in eliminating hematological malignant cell lines by natural killer cells].

The aim of this study was to clarify whether NKG2D plays an activating role in eliminating hematological malignant cells lines by natural killer (NK) cells. Several hematological malignant cell lines (K562, NB4, Kasumi-1 THP-1, MV-4-11, MOLT-4, Jurkat, RS4; 11, Raji) were used as target cells. The expression levels of major histocompatibility complex class I (MHC I)-related molecules A/B (MICA, MICB), whose corresponding ligand was NKG2D, were detected in target cells by flow cytometry. Firstly, the target cell lines were co-incubated with carboxyfluorescein succinimidyl ester (CFSE) for 30 min. In the meanwhile, NK92MI, a kind of NK cell line, was co-incubated respectively with isotype control antibody or blocking antibody, the latter could block NKG2D specifically. Then, NK92MI cells were co-cultured with different target cell lines. After incubation for 2 h, the apoptotic ratio of each target cell line was detected by flow cytometry. The results demonstrated that there was a significant reduction of the apoptotic ratio in Kasumi-1, an acute myeloid leukemia cell line, when NK92MI cells were incubated with NKG2D blocking antibody previously. In contrast, the apoptotic ratio of other cell lines varied minimally. It is concluded that NKG2D can activate NK cells through inducing cytotoxicity to certain target cells.

[Analysis of ITD characteristics in acute myeloid leukemia patients with FLT3-ITD positive].

The aim of this study was to analyze the FMS-like tyrosine kinase 3 internal tandem duplication (FLT3-ITD) allelic ratios (AR), number of ITD, ITD length and positions of ITD insertions in de novo acute myeloid leukemia (AML) patients with FLT3-ITD positive, and the relationship between mutant level and therapeutic efficacy. Genomic DNA was amplified by PCR, capillary electrophoresis was used to detect the ITD characteristics in 31 de novo AML patients, and DNA sequences analysis of FLT3-ITD(+) were performed in 13 patients. The results showed that the ratios of mutant to wild type FLT3 allele ranged from 0.01 to 2.8; 28 patients (90.32%) had a single ITD, the remaining 3 patients had more than one ITD; the ITD length ranged from 3 to 144 bp in all FLT3-ITD(+) patients. 13 sequence-analyzed patients, 4 patients were of pure duplications, and 2 patients had foreign bases inserted, and the other 7 patients were partial duplications. The ITD occurred in the regions from p.E573 to p.P606 of the FLT3 protein, with the majority clustered in a stretch between p.F590 and p.R595. The complete remission (CR) rate in AR < 0.5 patients (43.75%) were more prevalent as compared with AR ≥ 0.5 patients (16.67%) (p > 0.05). It is concluded that the ITD length and AR are vary widely. Some of the insertions are foreign bases, and all of the 13 sequences-analyzed ITD were concentrated on the juxtamembrane domain. The CR rate in patients of AR < 0.5 had no statistical significance compared with patients of AR ≥ 0.5.

[FMS-like tyrosine kinase 3 gene mutation in acute myeloid leukemia detected by denaturing PAGE and its clinical significance].

The aim of this study was to analyze the frequency of flt3 length mutation (flt3-LM) in de novo acute myeloid leukemia patients and the relationship between flt3-LM and chromosome alterations, FAB subgroups, as well as efficiency of therapy. Genomic DNA was amplified by PCR; 2% agarose gel or 8% denaturing PAGE were used to detect the length mutation of flt3 gene in 99 de novo acute myeloid leukemia patients; karyotyping in 72 AML patients was performed by G banding technique. The results showed that the flt3-LM was detected in 20.2% (20/99) patients by agarose gel electrophoresis, and in 29.9% (29/99) by denaturing PAGE. The flt3-LM was not detected in M(0) (only one patient was available), but flt3-LM occurrence in AML subtypes was as follow: in M(2) (9/30), M(3) (6/27), M(4) (4/14), M(5) (7/19), M(6) (3/8) respectively. flt3-LM in patients with normal karyotypes (39.13%) was more prevalent as compared with patients of abnormal karyotype (24.49%), but there was no statistical difference (p > 0.05). The complete remission (CR) rate in flt3-LM positive patients (36.36%) was lower than that in flt3-LM negative patients (62.75%) in the 73 patients (p < 0.05) whose karyotypic detection was performed. The distributions of flt3-LM were observed in 8 out of 40 CR patients, 8 out of 21 PR patients, and 6 out of 12 NR patients. It is concluded that the denaturing PAGE is more sensitive and reliable to detect the flt3-LM. The flt3 mutation represents a common genetic abnormality in AML patients, and the flt3-LM is associated with lower CR rate.

[Association of gene FN1 with pulmonary metastasis of human fibrosarcoma].

OBJECTIVE: To investigate the genes involved in pulmonary metastasis of human fibrosarcoma HT1080 cells in nude mice. METHODS: HT1080 cells were injected into the tail vein of BALB/ C nude mice. RNA samples were extracted from pulmonary metastatic tissues and normal control lung tissues, purified using Atlas Pure Total RNA labeling System (Clonetech Laboratories). cDNA probes labeled with 32P were prepared and hybridized to a cDNA membrane constructed with spots of 1176 human cancer related genes and radioactivities on the membrane were measeured by BAS 5000. The mRNA expression of gene FN1 was determined by real time RT-PCR using TaqMan methods. Furthermore, cells with FN1 expression were localized and obtained in situ in pulmonary metastatic foci by laser captured microdissection, and the FN1 expression was quantitated by real time RT-PCR. RESULTS: Of the total 1176 genes, 27 genes (2. 3%) revealed to be apparently up-regulated and 4 genes (0. 3% ) down-regulated. Real time RT-PCR analysis verified significant up-regulation of gene FN1. Laser captured microdissection/ real time RT-PCR analysis demonstrated up-regulated gene FN1 not in stroma cells but in tumor cell nests. CONCLUSION: Gene FN1 expression in fibrosarcoma HT1080 cells may be involved in pulmonary metastasis.

[Cyclosporine A based therapy for myelodysplastic syndrome].

To determine the efficacy and tolerance to cyclosporine A (CsA) based therapy in patients with myelodysplastic syndrome (MDS), 16 patients with MDS consisting of 10 refractory anemia (RA) and 6 refractory anemia with accessory blasts less than 10% (RAEB-1) were analyzed. Five patients had hypocellular bone marrows and 11 patients had normocellular or hypercellular marrows. The dose of CsA was 2.5-5.5 mg/(kg.d) for 2 weeks to 2 years (mean 8 months). Two out of 16 patients were treated with CsA alone, 14 patients were treated with CsA, recombinant human erythropoietin, androgens, 1, 25 dihydroxy vitamin D(3) or two or three of them combination with CsA. Treatment responses were classified according to the International Working Group (IWG) criteria as complete remission (CR), partial remission (PR), hematological improvement (HI) and no response (NR). Patients who obtained CR, PR or HI were defined as responders. The results showed that HI was observed in 12 patients, PR in 2 patients and NR in 2 patients. Total response rate was 87.5%. Response rates shown in neutrophil lineage, platelet and erythroid lineage were 83.3%, 66.7% and 60%, respectively; their shortest time required to obtain some hematologic improvement after initiation of CsA therapy was 2 weeks, 1 month and 1 month, respectively. Of 13 patients being transfusion-dependent before treatment, 3 patients did not need transfusion any more and 5 showed the reduced transfusion requirements after CsA therapy. In 10 patients with RA, 9 responded to CsA. Of 6 patients with RAEB, 1 patient had no response and died of RAEB-t and 5 patients had transient responses. One of the latter transformed to CMML and two relapsed. The total response rate decreased to 50% in the patients with CsA therapy lasting more than 3 months at the end of following-up. The adverse effects included hirsutism, hyperplastic gingiva, reversible hepatic and renal dysfunction. In conclusion, the usefulness of CsA based therapy for MDS-RA and RAEB-1 with any marrow cellularity is useful, the CsA dose of 3-5 mg/(kg.d) is safe and efficacious.

[Comparison of therapeutic effects of low-dose versus high-dose interferon alpha-2b treatment on chronic myelocytic leukemia: a prospective randomized study].

OBJECTIVE: To compare the therapeutic effects of low-dose and high-dose interferon alpha-2b (IFN) treatment on chronic myelocytic leukemia (CML). METHODS: A real-time quantitative reverse transcriptase PCR (RQ-PCR) method was established to detect the fusion gene bcr-abl expression, thereby studying the reduction of leukemic cells. Thirty newly diagnosed CML patients, 21 males and 9 females, aged 14 - 69, were treated with hydroxyurea to keep the white blood cell count less than 20 x 10(9)/L, and then randomized into 2 groups: high-dose IFN group receiving IFN alpha-2b 5MIU 6 times per week for 3 - 6 months and low-dose IFN group receiving IFN alpha-2b 3MIU every other day for 3 - 6 months. Bone marrow was collected every month to Real-time PCR was used to detect the expression of bcr-abl mRNA. Mononuclear cells were isolated and RNA was extracted to detect the expression of fusion gene bcr-abl and a control gene GAPDH. The results were reported as the number of bcr-abl copies/GAPDH copy. RESULTS: The established real-time quantitative PCR method could detect the bcr-abl molecules as low as 50 copies. The intra-assay coefficient of variation (CV) was less than 5% and the inter-assay CV was 5.13%. The median bcr-abl fusion gene expression level of 30 CML patients before IFN therapy was 0.098 (range: 0.010 - 5.799). The bcr-abl expression level decreased by 19.37% and 24.86% in the low-dose and high-dose IFN groups respectively after 3 months' therapy. No significant difference was observed between the two groups (P = 0.398). Relatively more side effects were observed in the high-dose IFN group than in low-dose group. CONCLUSION: RQ-PCR is a reliable method to monitor CML therapy by analyzing fusion gene bcr-abl expression. There is a difference in bcr-abl fusion gene expression levels among the newly diagnosed patients, and low-dose IFN is as effective as high-dose IFN in reducing bcr-abl expression but with less side effects.