Intracellular disposition of chitosan nanoparticles in macrophages: intracellular uptake, exocytosis, and intercellular transport.

Biodegradable nanomaterials have been widely used in numerous medical fields. To further improve such efforts, this study focused on the intracellular disposition of chitosan nanoparticles (CsNPs) in macrophages, a primary cell of the mononuclear phagocyte system (MPS). Such interactions with the MPS determine the nanoparticle retention time in the body and consequently play a significant role in their own clinical safety. In this study, various dye-labeled CsNPs (about 250 nm) were prepared, and a murine macrophage cell line (RAW 264.7) was selected as a model macrophage. The results showed two mechanisms of macrophage incorporation of CsNPs, ie, a clathrin-mediated endocytosis pathway (the primary) and phagocytosis. Following internalization, the particles partly dissociated in the cells, indicating cellular digestion of the nanoparticles. It was proved that, after intracellular uptake, a large proportion of CsNPs were exocytosed within 24 h; this excretion induced a decrease in fluorescence intensity in cells by 69%, with the remaining particles possessing difficulty being cleared. Exocytosis could be inhibited by both wortmannin and vacuolin-1, indicating that CsNP uptake was mediated by lysosomal and multivesicular body pathways, and after exocytosis, the reuptake of CsNPs by neighboring cells was verified by further experiments. This study, thus, elucidated the fate of CsNPs in macrophages as well as identified cellular disposition mechanisms, providing the basis for how CsNPs are recognized by the MPS; such information is crucial to numerous medical applications of CsNPs.

LC-MS/MS method for the simultaneous quantification of 11 compounds of Ginkgo biloba extract in lysates of mesangial cell cultured by high glucose.

The mesangial cell (MC) cultured with high glucose has been used to observe the protective effect of Ginkgo biloba extract (GBE) against diabetic nephropathy (DN), but the compounds interacting with cell are still unknown, which may be potential bioactive components. Based on this, the determination of GBE in MC lysates was proposed by high performance liquid chromatography tandem mass spectrometry (LC-MS/MS) in this study. The MC was cultured with normal or high glucose with GBE for 4, 8, 12, 16, 24 and 48h. The harvested cell was extracted with 40% acetic acid in water and further analyzed by LC-MS/MS. All the validation data including linearity, intra-day and inter-day precision, limit of detection and quantification, matrix effect, and stability were within the required limits. The validated method was successfully applied to quantify 11 compounds of GBE in cell lysates. The results showed that high glucose prolonged the peak time of all observed 11 compounds and peak concentrations of bilobalide, ginkgolide C, ginkgolide B, quercetin, luteolin, kaempferol, isorhamnetin and genkwanin in cell lysates, which hinted that these compounds may be the potential bioactive components of GBE with preventive effect against DN.

Screening for Potential Bioactive Components in Ginkgo biloba Extract by the Rat Renal Tubular Epithelial Cell Extraction and LC-MS/MS.

Rat renal tubular epithelial cell (RTEC) cultured with high glucose has been used to observe the protective effect of Ginkgo biloba extract (GBE) against diabetic nephropathy (DN). The compounds in GBE binding with cell membrane or entering into cell are still unknown, which may be potential bioactive components. In this paper, a powerful method for screening and analyzing the potential bioactive components from GBE was developed using cell extraction coupled with high performance liquid chromatography tandem mass spectrometry (LC-MS/MS). 8 prototype compounds and 5 metabolites were obtained, among which 6 prototype compounds and 1 metabolite were identified or tentatively characterized as rutin, bilobalide, ginkgolide B, ginkgolide C, genkwanin, apigenin and diosmetin by comparing their retention times and MS spectra with those of authentic standards or literature data. The 6 prototype compounds were further quantitatively analyzed using electrospray ionization in negative mode multiple reaction monitoring (MRM). The results showed that high glucose changed the Tmax, MRT(0-t), Cmax and AUC(0-t) of all observed compounds and decreased the t1/2 of genkwanin and apigenin, significantly. The overall findings indicate that 8 prototype compounds may be the potential bioactive components of GBE with preventive effect against DN and the method of RTEC extraction coupled with LC-MS/MS technology screening method we developed is a feasible, rapid, and useful tool for screening and analyzing potential bioactive components.

Target cell extraction coupled with LC-MS/MS analysis for screening potential bioactive components in Ginkgo biloba extract with preventive effect against diabetic nephropathy.

A rapid and useful approach for screening potential bioactive components in Ginkgo biloba extract (GBE) with preventive effect against diabetic nephropathy (DN) was developed using mesangial cells extraction coupled with high-performance liquid chromatography tandem mass spectrometry (LC-MS/MS) analysis. Mesangial cells were first divided into two groups according to their treatments with high glucose or high glucose plus GBE. After incubation for 4, 8, 12, 16, 24 and 48 h, the cells were harvested and extracted with 40% acetic acid in water before LC-MS/MS analysis. Then, 19 compounds and five metabolites were found to selectively combine with mesangial cells. Notably, compounds including quercetin and rutin were identified or tentatively characterized according to the results of retention time and MS spectra, which is highly consistent with our previous reports that quercetin and rutin are potent protective agents against glomerulosclerosis in DN. Therefore, all these results indicate that target cell extraction coupled with LC-MS/MS analysis can be successfully applied for predicting the bioactive components in GBE with preventive effect against DN.

[The clinical and experimental analyses of a multiple myeloma patient with derivative der(Y;1)].

OBJECTIVE: To investigate the clinical significance of a rare chromosome abnormality der(Y)t(Y;1) in a patient with multiple myeloma (MM). METHODS: The chromosome spread was prepared after 24 h culture of bone marrow. G-banding technique was used to analyze the karyotype. Fluorescence in situ hybridization (FISH) was performed to ascertain the origin of abnormal chromosome detected by conventional karyotypic analysis. Flow cytometry was used to detect the expression of the CD38/CD138/ZAP70. Immunoelectrophore was applied to identify the type of immunoglobulin. RESULTS: A complex pattern of chromosome rearrangement was observed: 92,XXYY[3]/49,X,der(Y)t(Y;1)(q12;q21),t(11;14)(q13;q32),+18,+20,+21[47]/49,X,idem,del(13q22),ace[1]/98,XX,der(Y)t(Y;1) x 2,+18,+18,+20,+20,+21,+21[10]/46,XY[19]. The result was confirmed by metaphase-FISH. The type of immunoglobulin was IgD with the level of 6.24g/L. The CD38/CD138 was positive but ZAP70 was negative. CONCLUSION: Structural abnormality of chromosome Y is rare in blood malignancy. Most of them were described in myelodysplastic syndrome or myeloproliferative disorders. It is the first report of der(Y)t(Y;1) abnormality in multiple myeloma.

[Analysis of variant translocation der ins (17; 15) in patient with APL by G-banding technique and interphase fluorescence in situ hybridization].

To investigate the biological characteristics of the variant translocation der ins (17;15) in a patient with acute promyelocytic leukemia (APL), the conventional G-banding technique, interphase fluorescence in situ hybridization (int-FISH), RT-PCR, gene scanning, gene sequence and flow cytometry were performed. The results indicated that the variant translocation der ins (17, 15) observed by G banding technique was a rare type, the int-FISH assay by using dual-color pml/raralpha fusion probes confirmed the cytogenetic findings. The detection results of other molecular methods demonstrated the existence of the whole pml/raralpha fusion gene, while this case had insertion variant translocation. This patient got complete remission by using combined chemotherapy, and survives with continuous complete remission during following up for 1 year. In conclusion, the variant translocation der ins (17; 15) is rare type in APL, its incidence is lower, several signal types in detection of int-FISH were observed and the combination chemotherapy for this patient showed more obvious efficacy.

[FIP1L1/PDGFRalpha fusion gene-negative chronic eosinophilic leukemia with t(5;12)(q31;p13): a case report and review of literatures].

OBJECTIVE: To deepen the understanding of chronic eosinophilic leukemia (CEL). METHODS: The course of diagnosis and treatment in a case of FIP1L1/PDGFRalpha fusion gene negative CEL was reported. Flow cytometry was used to analyze the immunophenotype of the cells in peripheral blood and pleural fluid. Karyotype was analyzed with G-banding. The expression of FIP1L1/PDGFRalpha fusion gene was detected by RT-PCR technique. Routine pathological examination of the tissues from bone marrow, lung and spleen were performed. RESULT: A sixteen-year-old girl had severe anemia, fever, splenomegaly, thrombocytopenia and dominant hypereosinophilia lasting for 22 months. Trephine biopsy showed a hypercellular marrow with eosinophilic proliferation and moderate reticular fibrosis. Eosinophilic infiltration was found in lung and spleen and embolism was also found in spleen. She had a clonal chromosomal abnormality t(5;12)(q31;p13). The expression of FIP1L1/PDGFRalpha was negative. An abnormal clone of T cells expressing CD(3)(-), CD(4)(-), CD(8)(+) was found in peripheral blood and pleural fluid, in which the clonal T cell accounted for 5.43% and 1.66% of the total lymphocytes respectively. The patient was refractory to treatment with hydroxyurea, prednisone and interferon alpha. She had poor response to a combination of therapy with low dose cytosine arabinoside, mitoxantrone, vincristine, cyclophosphamide, methotrexate and prednisone. She did not respond to imatinib and died of multiple organ failure. CONCLUSION: The present case fulfilled the WHO diagnostic criteria of FIP1L1/PDGFRalpha(-) CEL which did not respond to routine treatment and imatinib. Allogenic stem cell transplantation should be considered as early as possible in this case. It is noteworthy that clonal CD(3)(-), CD(4)(-), CD(8)(+)T-cell abnormality is related to the pathogenesis of CEL.

[Characteristics of cases with chromosome 3q21q26 aberrations].

To investigate the cytogenetic and clinical characteristics of inv(3q) (q21q26) and t(3;3) (q21; q26) aberrations as well as prognosis, cases were collected and chromosome specimens of bone marrow cells were prepared by 24-hour culture, while G-banding technique was used to perform karyotyping. The results showed that the simple inv(3q) and t(3; 3) aberrations were rare, they commonly combined with other chromosome aberrations such as -7/7q- and t (9; 22). The involved diseases included myelodysplastic syndromes, acute myeloid leukemia and chronic myelogenous leukemia in blast crisis. Out of 24 patients, 2 patients diagnosed with M(5) subtype did not achieve complete remission after multiple chemotherapy; 2 patients received allogenic stem cell transplantation relapsed. It is concluded that 3q21q26 aberration commonly combined with chromosome aberration 7/7q-, for these patients the efficacy of chemical therapy is poor, the efficacy of bone marrow transplant is too poor, these patients with inv(3q) and t(3; 3) aberrations have poor prognosis and short overall survival.

[The prognostic implications of secondary chromosomal aberrations in Philadelphia chromosome-positive chronic myeloid leukemia patients after imatinib mesylate treatment].

OBJECTIVE: To investigate the change of Philadelphia chromosome-positive clone with secondary chromosomal aberrations after imatinib mesylate (IM) treatment in patients with chronic myeloid leukemia (CML) and its relation with prognosis. METHODS: 37 cases of CML in accelerated phase and blastic phase were collected and chromosome specimens of bone marrow cells were prepared by with 24-hour culture. G-banding technique was used for karyotyping. RESULTS: The major secondary chromosomal aberrations were double Ph, +8, and i (17q); the minor ones were -Y, chromosome 17 abnormalities other than i (17q) and inv (3q). The percentage of Philadelphia chromosome-positive clone with secondary chromosomal aberrations showed the following 4 types of change; amplification, no change, decrease and complete remission after treatment with IM. 2 of the 24 cases in CML in accelerated phase gained complete cytogenetic response (CCyR) and 2 of the 13 in blastic phase did so. The groups with amplification and no change showed significantly shorter overall survival and progression free survival than the groups with decrease and complete remission. CONCLUSION: The percentage of Philadelphia chromosome-positive clone with secondary chromosomal aberrations may drop in some CML patients after IM treatment and the patients may gain CCyR with accompanied prolonged survival.

[Cytogenetic study on eosinophilia].

The aim of study was to investigate the importance of chromosome aberration in differential diagnosis of eosinophilia and the chromosomal aberrations involved in patients with clonal eosinophilia. 65 cases of eosinophilia were collected and chromosome specimens of bone marrow cells were prepared by 24-hour culture, and G-banding technique was used for karyotyping. The results showed that out of 65 cases, chromosome 16 inversion was detected in 9 patients suspected as M(4Eo), and among the other 56 cases, 5 were detected with chromosomal aberrations (8.9%). Combining clinical, hematological and cytogenetical data, the 5 patients were diagnosed as acute myeloid leukemia with eosinophilia, chronic eosinophilic leukemia, 8p11 myeloproliferative syndrome, chronic myeloid leukemia in acute phase and acute myeloid leukemia-M(4Eo) respectively. The detected chromosomal aberrations were +14, t (5; 12) (q31; p13), t (8; 9) (p11; q32), t (9; 22) (q34; q11) and inv (16) (p13 q22). In conclusion, cytogenetical detection is very important in differential diagnosis of clonal eosinophilic disorders and chronic eosinophilic leukemia, which is suggested to be done routinely in clinic.

[Cytogenetic study of multiple myeloma].

To investigate the cytogenetic characteristics of multiple myeloma and its relationship with clinical prognosis, 68 cases were collected and chromosome specimens of bone marrow cells were prepared by 24-hour culture, and G-banding technique was used for karyotype analysis. The results showed that the detected chromosome aberration rate was 19.1% (13/68). The abnormal clones existed mosaically with normal clones. Numerous aberrations were manifested by aneuploidy, mainly by hyperdiploidy or hypodiploidy. Structural aberrations involved t (11; 14), chromosome 1 and various kinds of marker chromosomes. Cases which had very complex aberrations revealed poor prognosis. It is concluded that chromosome complex aberration is the mainly cytogenetic characteristics of multiple myeloma, and multiple numerous and structural aberrations are involved. Cytogenetic detection should be performed both at diagnosis and at disease progression so as to evaluate prognosis.

[The efficacy of imatinib mesylate for 124 patients with chronic myeloid leukemia in accelerated and blastic phase].

OBJECTIVES: To evaluate the efficacy and safety of imatinib mesylate (imatinib) for patients with Philadelphia chromosome-positive (Ph+ ) chronic myeloid leukemia (CML) in accelerated and blastic phase. METHODS: Seventy-five Ph+ CML patients in accelerated phase and 49 in blastic phase were treated with 400 mg or 600 mg of imatinib once daily. RESULTS: For patients in accelerated phase, the cumulative hematological response (HR) rate was 93.3%, including complete HR (CHR) rate 85.3%, and returning to chronic phase (RCP) rate 8% in a median follow-up of 23.0 (1.0 -64.0 ) months. Cumulative major cytogenetic response (MCyR) rate was 33.0%, and complete cytogenetic response (CCyR) rate 28.0%. For patients with CCyR, the major molecular response (MMoR) rate was 47.6%. The estimated 4-year progression-free survival (PFS) rate and overall survival (OS) rate were 48.2% and 52.2% in patients with HR, respectively. Severe leukocytopenia, anemia and thrombocytopenia occurred in 37.3%, 34.6% and 45.3% of all patients, respectively. For patients in blastic phase, the cumulative HR rate was 63.3%, including CHR rate 44.9%, and RCP rate 18.4% in a median follow-up of 4.5 (0.3 -63.0) months. Cumulative MCyR rate and CCyR rate were both 12.2%. For patients with CCyR, the MMoR rate was 33.3%. For patients with HR, the estimated 1-year/2-year PFS and OS rates were 32.8%/15.8% and 46.0%/ 21.0% respectively. Severe leukocytopenia, anemia and thrombocytopenia occurred in 75.5%, 71.4% and 73.5% of all patients, respectively. CONCLUSIONS: The efficiency of imatinib was decreasing, and severer hematological toxicities increasing with the disease progressing in patients with Ph+ CML. Imatinib improves progression-free survival significantly in most patients in accelerated phase, particularly in those with continuous CCyR or MMoR. The response duration in majority of blastic phase patients is short, and the relapse rate is high.

[Studies of treatment strategy and prognosis on acute myeloid leukemia with chromosome 8 and 21 translocation].

OBJECTIVE: To investigate the relationship between the biological features and the treatment efficacy and prognosis in acute myeloid leukemia subtype M2 (AML-M2) patients with chromosome 8 and 21 translocation. METHODS: By using Cox regression model and Kaplan-Meier analyses, prognostic factors in 54 cases of de novo adult AML with t(8;21) in our institute from 1990 to 2003 were retrospectively analyzed. RESULT: The complete remission (CR) rates were 81.9% for all M2 patients, 82.4% for patients with normal karyotype, 88.5% for patients with t(8;21) [P > 0.05 for normal karyotype vs t(8;21)], 100.0% for 28 patients with t(8;21) alone and 75.0% for 24 patients with additional chromosome abnormalities (P < 0.01). The actuarial 3 year overall survival(OS) was 26% for M2 patients with normal karyotype, 25% for patients with t(8;21) [P > 0.05 for normal karyotype vs t(8;21)], in whole t(8;21) group, 46.4% for patients with t(8;21) alone and 0% for patients with additional chromosome abnormalities (P < 0.01). Multivariate analysis of prognostic factors showed that chromosome abnormalities besides t(8;21) was the only factor affecting CR, disease-free survival (DFS) and OS. DFS of allogeneic hematopoietic stem cell transplantation (HSCT) and intermediate-dose cytarabine/high dose cytarabine (IDAC) groups were better than the group received routine dose cytarabine as postremission therapy (P < 0.01). CONCLUSION: AML with t(8;21) is not a single defined AML subset, and patients with additional chromosome abnormalities have a worse prognosis. HSCT and IDAC could improve the outcome. HSCT is the best choice for patients with high risks, especially with additional chromosome abnormalities.

Characteristics and prognostic factors of acute myeloid leukemia with t (8; 21) (q22; q22).

The translocation t (8; 21) (q22; q22) frequently associated with additional chromosomal aberrations is one of the most recurrent chromosomal abnormalities in AML. Clinically, this type of AML usually shows some specific characteristics and has a good response to chemotherapy with a high remission rate and a relatively long median survival. On the other hand, some reports also showed poor prognosis in AML patients with t (8; 21), and the associated bad-prognosis factors have not been strongly established to date. To investigate this issue and to further identify the related characteristics of t (8; 21) AML in China, 75 Chinese AML patients with t (8; 21) were retrospectively analyzed. They comprised 68 cases of M(2), five of M(4) and two of M(5) according to FAB classification. The results indicated that Auer rods were observed in 39 patients (52%) and marrow eosinophilia was detected in only 5 patients (6.7%). These patients showed high level of HLA-DR and CD34 expression, while CD19 was detected in only 13 patients (20.9%). Cytogenetically, 62.5% cases had additional chromosomal abnormalities, and the main associated recurrent additional abnormalities were loss of a sex chromosome (LOS), trisomy 4, del (9q) and trisomy 8. After conventional induction therapy, 62 patients attained complete remission (CR) resulting in the CR rate 82.7%. With a follow-up of 1 to 96 months, 19 cases relapsed at a median time of 10.5 months (range 3 to 42 months). The median overall survival was 20 months, and the estimated 5-year overall survival (OS) rate was 32.3%. In multivariate analyses of prognostic factors, karyotype, extramedullary leukemia, age and post-remission therapy were of prognostic value for OS. Patients with additional chromosomal anomalies had shorter survival compared to those with t (8; 21) only (P = 0.019), no matter which kind of additional karyotype it was. Extramedullary leukemia was an adverse prognostic factor (P = 0.012). Patients aged 15 years or less had a longer survival than those aged more than 15 years (P = 0.045). Patients accepted HSCT in post-remission therapy had better outcome compared to those with chemotherapy only. It is concluded that Chinese AML patients with t (8; 21) had some different characteristics as compared with patients from other countries, a relatively poor outcome was observed in our patients, especially in those with extramedullary leukemia or additional chromosomal abnormalities. HSCT should be recommended to t (8; 21) AML in China, especially to those with adverse prognostic factors.

[Treatment of refractory and relapsed acute lymphocytic leukemia in adults].

OBJECTIVE: To analyze on the efficacy and toxicity of fludarabine and teniposide + mitoxantrone (MIT) regimens on treating refractory and relapsed acute lymphocytic leukemia in adult patients. METHODS: Teniposide 100 mg/d, 5-7 d, MIT 10 mg/d, 2 d and fludarabine regimens [Flu 30 mg/(m(2) . d), 3- 5 d, Cytarabine (Ara-c )1-2 g/(m(2) . d), 5 d; Flu 50 mg/d, 5 d, Ara-c 200 mg/d, 5 d, MIT 4 mg/d, 4 d] were used to treat 42 cases of adults with refractory and relapsed acute lymphocytic leukemia(ALL). G-CSF 5 microg/(kg . d) were used when WBC<1.0 x 10(9)/L. RESULTS: In both the regimens fludarabine and VM (teniposide + MIT), the complete remission (CR) rate was 45% versus 31.8% (P>0.05); the median neutropenia began 6 days after the regimens arresting and lasting 10 versus 7.5 days, P>0.05; thrombocytopenia begin at time of 10 versus 6.5 days (P<0.05) after the regimens arresting and lasting 6 versus 10 days (P>0.05). Fludarabine regimen had less non-haematological toxic effect than that of VM. CONCLUSION: Compared with VM, Fludarabine regimen was a very effective alternative treatment for CR induction in adult patients with refractory and relapsed ALL and low toxicity.

[Clinical and cytogenetical study on subacute myeloid leukemia in myelodysplastic syndromes].

OBJECTIVE: To discuss from the clinical and cytogenetic aspect that part of patients now diagnosed as myelodysplastic syndromes (MDS) could be diagnosed early as leukemia and be classified as subacute myeloid leukemia (Sub-AML). METHODS: Totally 173 patients diagnosed as MDS according to FAB or WHO criteria with complete clinical and cytogenetical data were included in this research. Among them 42 had +8 chromosome aberration, 16 had -7/7q-, and 55 had normal karyotypes and more than 0.10 blast cells in the bone marrow. Short term culture and G-banding techniques and in some specimens fluorescence in situ hybridization (FISH) method were used to do chromosome analysis. RESULTS: Among the detected chromosome aberrations, +8 was the most frequent (42.8%) and then -7/7q-(15.0%); 42 patients with +8 had median blast cell count of 0.08, within a median of 18 months follow-up period 40.0% of the patients evolved to frank leukemia (FL) and the median overall survival was 20 months. 16 patients with -7/7q- had higher blast cell count of 0.135; 43.8% of them developed into FL and the median overall survival was only 10 months within a 20-month follow-up period. 55 patients had normal karyotype but a median blast cell count of 0.148; 52.7% of them patients evolved to FL and the median overall survival was 34 months. CONCLUSIONS: Both the +8 and -7/7q- groups have malignant leukemic cell clone, and run a subacute and progressive clinical course; it is suggested they might be classified into Sub-AML. We should keep close watch on the patients who have normal karyotype yet more than 0.10 blast cells, part of whom might suffer from early Sub-AML.

[Analysis of characteristics of 72 cases of t(8;21) acute myeloid leukemia].

OBJECTIVE: To investigate the laboratorial and clinical characteristics of t(8;21) AML, and to compare the differences between patients with additional chromosomal abnormalities and those without additional aberrations. METHODS: Seventy-two cases of t(8;21) AML were analyzed retrospectively, including features of morphology, initial blood cells, cytogenetic G-banding karyotype, immunophenotype, AML1/ETO fusion gene and clinical outcome. In order to compare the characteristics of patients with additional chromosomal abnormalities with those with t(8;21) alone, these patients were divided into two groups according to their karyotype as follows: Group A included patients with t(8;21) alone; Group B included patients with additional aberrations. RESULTS: According to FAB classification, there were 65 cases of M2, 5 cases of M4 and 2 of M5. Cytogenetically, 45 cases (62.5%) were accompanied by additional chromosomal abnormalities. The main additional aberrations included -Y, +4, del(9q). There were no obvious differences between these two groups in their features of age distribution, bone marrow blast cells, Auer rods, eosinophilia, immunophenotype, as well as central nervous system leukemia occurrence and complete remission rate of induction, but a male prevalence and a lower initial WBC were observed in Group B. With a follow up of 1-96 months, the median survival time was much longer in group A (65 months) compared with group B (12 months), but there was no difference between the four main subgroups of group B. The estimated 3-year survival was (63.9+/-11.2)% in group A compared with (20.9+/-9.2)% in group B. CONCLUSION: Additional chromosomal abnormality is an adverse factor for prognosis of t(8;21) AML. The median survival time of patients with additional aberrations was much shorter than that of those without.

[Cytogenetic and clinical study of Philadelphia chromosome positive adult acute leukemia].

To explore the cytogenetics and related clinical characteristics of adult acute leukemia with Philadelphia chromosome positive (Ph(+)AL), MIC classification by morphology, immunology and cytogenetics was used to retrospectively study 79 patients with Ph(+)AL hospitalized in the Institute of Hematology, People Hospital in Beijing from October 1991 to September 2003. The results showed that 6.9% cases were diagnosed as Ph(+)AL and classified into three subtypes: acute lymphoblastic leukemia (Ph(+)ALL) in 56 patients (18%), acute myeloid leukemia (Ph(+)AML) in 10 patients (1.2%) and mixed acute leukemia (Ph(+)MAL) in 13 patients. B-cell antigen expression was found in 52 out of 56 patients with Ph(+)ALL. 54.4% (43/79) patients had additional chromosome abnormalities including chromosome 7, double Ph and plus 8, etc. Complete remission (CR) rate of Ph(+)ALL and Ph(+)MAL was 57.0%, none of Ph(+)AML achieved CR. Median overall survival of Ph(+)ALL, Ph(+)MAL and Ph(+)AML were 10, 10 and 2.5 months respectively. It is concluded that Ph(+)AL has highly heterogeneity involving various differentiated stages of immature leukemic cells. Since the poor prognosis associated with this kind of AL, early diagnosis with MIC classification is a prerequisite to take more effective conditioning regimen and prospectively consideration of allogeneic stem cell transplantation to improve prognosis.

[Monitoring bcr/abl mRNA levels in imatinib mesylate treated chronic myeloid leukemia patients by real-time quantitative RT-PCR].

OBJECTIVE: To quantify bone marrow bcr/abl mRNA levels in imatinib mesylate treated Ph chromosome positive chronic myeloid leukemia (CML) patients. METHODS: Serial monitoring of bcr/abl mRNA levels by real-time quantitative RT-PCR technique (RQ-PCR) was performed in 34 cases (120 samples) of CML treated with imatinib mesylate. All the patients were IFNalpha based treatment failure before enrolled in this study and the percentage of Ph(+) bone marrow cells were over 95%. RESULTS: The sensitivity of RQ-PCR was 10 pg RNA, with both coefficients of interassay and intraassay variation below 5% for standard samples. The median bcr/abl mRNA level of 10 patients' samples pre imatinib treatment was 5.79% with marked variation (0.24%-60.90%). In 72 samples post imatinib treatment, which the rates of Ph(+) cells [Ph(+)%] were between 0 and 94%, the mRNA level well correlated with Ph(+)% (r = 0.82, P < 0.001). The mRNA levels of 7 patients who achieved complete cytogenetic response (CCyR) within 12 months decreased markedly, the levels of 6 analysable patients decreased by 65.9% - 98.8% after 3 months'treatment accordingly. The level further decreased to 0 after achieving CCyR. For 4 patients who achieved major cytogenetic response (Ph(+) cells < 35%) later than 12 months, the mRNA levels decreased slowly. The levels of 3 analysable patients on 3 month therapy decreased by 2.5%, 18.5% and 61.6% compared with that before treatment. Out of 5 patients in chronic phase without cytogenetic response, 1 decreased, 2 increased gradually and 2 had no change. In 4 disease progression patients, the levels increased stepwise. CONCLUSIONS: Serial quantifications of bcr/abl mRNA levels are necessary for imatinib treated patients, and are more informative than a single detection. A sharp decline of bcr/abl mRNA levels after the treatment implies a promise of CCyR.

[Clonal evolution of abnormal Philadelphia chromosome-negative cells after imatinib mesylate therapy in patients with Philadelphia chromosome-positive chronic myelogenous leukemia].

OBJECTIVE: To investigate clonal evolution of abnormal Philadelphia chromosome-negative cells (Ph- CE) after imatinib mesylate therapy in patients with Philadelphia chromosome-positive chronic myelogenous leukemia (Ph+ CML). METHODS: Bone marrow cells G-banding karyotype was evaluated every 3 months in 100 patients with Ph+ CML after achieving hematologic responses on the course of imatinib therapy. There were 54 patients in chronic phase (CP), 37 in accelerated phase (AP) and 9 in blast phase (BP). RESULTS: After a median follow-up of 32 months (ranged 25-34 months), 11 patients, including 5 cases in CP, 5 in AP and 1 in BP, developed transient, interrupted or continuous Ph- CE after 3 - 29 months on imatinib therapy. Ph- CE emerged at the beginning of Ph+ cells decreasing or after Ph+ cells disappearing. The proportion of Ph- CE, was negatively correlated with the proportion of Ph+ cells (P < 0.05). Ph- CE commonly included +8 (45.5%) and +Y (27.3%). Five patients had additional cytogenetic abnormalities besides Ph+ in Ph- CE. Seven of the patients with Ph- CE achieved a major cytogenetic response while 9 of them achieved a complete hematologic response. One patient with Ph- CE in AP progressed to BP 20 months after the initiation of the therapy while the rests remained in hematologic or cytogenetic responses. CONCLUSION: Ph- CE occurred in about 11% of the patients with Ph+ CML who achieved major or minor cytogenetic responses on imatinib therapy. After a median follow-up of more than 2 years, most of the patients with Ph- CE were in a stable status with no disease progression.