The Clinical And Biological Studies Of Myelogenous Hematologic Malignancies

1. The clinical and biological studies of myelodysplastic syndromesObjectives①To analyse systematically the clinical and laboratorial characteristics of 1105 myelodysplastic syndrome patients who received conventional cytogenetics (CC) analysis in our laboratory from 1984 to 2009 and to reveal the unique features of MDS patient in our area.②To compare the difference of CC and panel ?uorescence in situ hybridization (FISH) in detection of frequent cytogenetic abnormalities of MDS patients, and to explore their diagnostic for MDS .Methods①R HG banding was employed for karyotypic analysis of 1105 MDS patients; All patients were classified according to the FAB proposals; in which, 745 cases were reclassified according to the WHO (2008) proposals; and 303 patients'outcomes were evaluated according to the International Prognostic Scoring System and WHO classi?cation–based Prognostic Scoring System (WPSS).②211 MDS patients were chosen randomly in all MDS patients mentioned above. A panel FISH probes, including 5q31/5p15.2, 7q31/cen7, Cen8, 17p13.1, 20q12 and Yq12, were used to detect the common chromosome abnormalities in bone marrow cells of these patients.Results According to the FAB group criteria, 1105 MDS patients were classified as follows: 543 (49.1%) patients with RA, 71 (6.4%) patients with RARS, 334 (30.2%) patients with RAEB, 116 (10.5%) patients with RAEB t, and 41 (3.7%) patients with CMML. The ratio of male and female was 1.55, and the median age was 48 years old (range, 2 89 years old). 42.6% patients had clonal chromosome abnormalities, and in which the rate of abnormalities was higher in RAEB than that in other subtypes. According to IPSS cytogenetic risk categories, IPSS good , intermediate and poor risk cytogenetics were 699 cases (63.3%), 238 cases (21.5%) and 168 (15.2%), respectively. The proportion of poor cytogenetics was higher in FAB subgroups with more than 5% blasts than that in subgroups with less than 5% blasts. Remarkably, the subgroup with RA had a longer median survival duration than the subgroup with RAS (P<0.05), and the median survival duration of the subtypes with more than 5% blasts is shorter than that of those subtypes with less than 5% blasts (P<0.05). But survival time differences between RAEB and RAEB t were not statistically significant (P>0.05)In adopting the WHO (2008) proposals, 745 patients remained classifiable, The new distribution included 132 patients (17.6%) with RA/RN/RT/RCUD, 28 patients (3.8%) with RARS, 147 patient (19.7%) with RCMD, 146 patients (19.6%) with RAEB 1, 150 patients (20.1%) with RAEB 2, 139 patients (18.7%) with MDS u, and 4 patient (0.6%) with 5q syndrome. The count of 5q syndrome patients in our study was far less than western report but similar as that of Asian other reports. The male was still domination, and median age was 50 years old (range: 6 89 year old). In all subgroups, the median hemoglobin level of RA/RN/RT/RCUD was higher than that of other subgroups, the median platelet count of RARS was more than that of others and the neutrophil count of RA/RN/RT/RCUD and RARS were less than that of others. The rate of clonal chromosome abnormalities was 41.9%, and the RAEB 1 had higher rate of abnormalities than other subgroup. IPSS good , intermediate and poor risk cytogenetics were 487 cases (64.2%), 156 cases (20.9%) and 111 cases (14.9%), respectively. The median survival duration of RCMD, RARS, RAEB 1 and RAEB 2 was 53.1 months, 40.2 months, 19.7 months and 17 months, respectively. No significant differences were observed between RA/RN/RT/RCUD, RCMD, RARS, 5q syndrome and MDS U, also, the median survival duration of RAEB I and RAEB II had no statistical difference(P>0.05). A difference with borderline significance was seen between the subtypes with more than 5% blasts and those subtypes with less than 5% blasts(P<0.05).CC analyses were performed on all 1105 MDS patients. A systematic documentation of cytogenetic abnormalities was performed on every patient. The abnormalities of chromosome 8 were most frequent, and then the abnormalities of chromosome 5, chromosome 7 and chromosome 20 were followed; The trisomy had the highest occurrence (occurring in 39.7% cases), the following were deletion of long or short arm (mainly long arm) (39.3%) and monosomy (22.5%). About 28 common chromosome abnormalities could be observed, which were +8(29.7%), 7/del(7q)(15.9%), del(20q)(12.5%), del(5q)( 11.7%) and etc. Comparing with western reports, the chromosome abnormalities in our study showed unique features as follows: a. isolate +8 was the most frequent in our study but isolate del(5q) was the common in western countries. b. The complex chromosome abnormalities containing 7/del(7q) was more than isolate 7/del(7q) in our study, on the contrary, the isolate 7/del(7q) was more than the complex chromosome abnormalities containing 7/del(7q) in the western report. c. the frequency of del(20q) in our study was far more than that in western reports. d. the del(5q) were chiefly on complex karyotypes or isolate respectively in our study and western reports. e. the rate of–Y was lower than that in western reports. f. a new chromosome abnormalities i(20q ) related to MDS was discovered in our study.According to IPSS, the most frequent was intermediate 1 risk group (47.9), and the low risk group was 8.9%, the intermediate 2 risk group was 26.7%, the high risk group was 16.5%. The median survival duration of intermediate 2 risk group and high risk group were 29.1 months and 12.4 months respectively. In adopting WPSS, 34 patients (11.2%) were in very low risk, 50 patients (16.5%) were in low risk, 46 patients (15.2%) were in intermediate risk, 82 patients (27.1%) were in high risk, and 28 patients (9.2%) were in very high risk. the median survival was 53.1 months for low risk group, 38.3 month for the intermediate risk group, 21.5 months for the high risk group and 10.3 months for the very high risk group. The higher risk strati?cation of IPSS and WPSS are classified, the poorer prognostic will be.Comparison with the results of panel FISH and CC, all specific chromosome abnormalities covered by panel probes were detected in 54 cases (24.4%) by panel FISH; panel FISH showed negative results in 22 cases (10.4%) whose chromosome abnormalities were out of the covered region of panel probe; In 4 cases (1.8%) with normal karyotypes and 3 cases (1.4%) with complex karyotypes detected by CC, the panel FISH revealed the chromosome abnormalities missed by CC.Conclusion①In our study, the MDS patients showed the unique clinical and biological features compared with western MDS patients. We found the 5% blast in bone marrow is a critical point, it has important prognostic significant; the poor cytogentic risk is related to poor outcome of MDS patients and WPSS is a powerful tool in MDS survival analysis.②CC is a basic examination of MDS patients; the panel FISH can detected common chromosome abnormalities accurately and sensitively. For those cases without metaphases, with poor metaphases or cryptic abnormalities, the panel FISH could improve detection rate of chromosome abnormalities. 2.A clinical and laboratory study of acute leukemia with t(16;21)(p11;q22)/FUS- ERG fusionObjectives①To explore the clinical, immunophenotype, cytogenetic and molecular genetic features of acute leukemia patients with t(16;21)(p11;q22)/FUS ERG positive.②To establish a novel human myeloid leukemic cell line and analyzed its biologic characterizes.Methods①23 acute leukemia patients with t(16;21)(p11;q22)/FUS-ERG fusion were collected in our study. And then, we used R banding technique to analyze karyotypes, PT PCR to detected the FUS ERG fusion gene transcript, and flow cytometry to analyze the immunophenotype. The clinical features and long term follow up of these patients were analyzed.②Mononuclear cells were isolated from the bone marrow of an acute myeloid leukemia patient with t(16;21)(p11;q22)/FUS ERG positive at his relapse, and were cultured in a 24 well cell culture cluster at a density of 1×106/ml in Iscove's Modified Dulbecco's Medium (IMDM) supplemented with 20% heat inactivated fetal calf serum (FCS) at 37℃in a 5% CO2 humidified in an incubator. JIH 4 cells were performed the characteristics as follows: morphological,cytochemical, cytogenetic and surface makers analysis, reverse transcription polymerase chain reaction (PT PCR) analysis, multiple fluorescence in situ hybridization (M FISH), growth curve analysis, detection of Epstein Barr virus (EBV) and mycoplasma, authentication of the cell line and clonogenic assay, tumorigenicity in mice and gene mutation analysis.Results①The incidence rate of acute leukemia patients with t(16;21)(p11;q22)/FUS ERG positive in acute leukemia was 4.5‰. These patients had balance sex ratio ( male/female was 12/11). Their median age was 24 years old (range from 7 to 77 years old). Of them, 1 case was diagnosed as acute lymphoblastic leukemia (ALL) and the others were acute myeloid leukemia (AML), in which M5 was domination; 2 patients were normal karyotypes with FUS ERG fusion gene positive, and others accompany with t(16;21)(p11;q22). In 19 cases detected by flow cytometry, 17 cases were myeloid phenotype, 1 case was myeloid and monocytic phenotype, and 1 case was B phenotype. ALL 13 cases detected by RT PCR were FUS ERG fusion gene positive, and they had three chimeric transcripts (type A, B and C) with the type B dominationally. Only 50% of them achieved the complete remission in first chemiotherapy, the 53.8% of them relapsed in two years, their relapsed free survival time were from 7 months to 15 months, and their median overall survival time was 8 months. Their overall survival time were shorter than that of patients with normal karyotype, simple t(15;17)(q22;q11), simple t(8;21)(q22;q22), and were similar as that of patients with complex karyotype.②One and a half months after cultured, the cultured cells showed a slow but stable proliferation. The cells started to proliferate rapidly after 3 month. Till now, the cells have been cultured in IMDM without any cytokines for more than 9 months. After suffering frozen and thawed repeatedly, it still successfully expands in culture. Meanwhile, it showed negative result of EBV and mycoplasma detection, and the complete correspondence with primary leukemic cells by STR PCR. Thus, it has been judged as a continuous cell line and designated as JIH 4. The doubling time of JIH 4 cells was 84.4 hours. The morphology of JIH 4 cells showed the large and range nucleus, with fine nuclear chromatin, one to two nucleoli and basophilic and fine cytoplasm. They strongly expressed CD13, CD33, CD56, CD56/CD16, and CD34 but negative for B and T lymphocytic lineage antigen, so, it is an AML cell line with NK related antigen expressed. The JIH 4 cell line showed the simple translocation between chromosome 16 and chromosome 21 by RHG and M FISH, and expressed the FUS/TLS-ERG fusion gene transcript with only one type transcript– type B. And, the JIH 4 can form the colony in semi solid methylcellulose clonogenic assay, had tumorigenicity in nude mice, and had no gene mutation of Kit, FLT3, JAK2, NPM, CEBPA, RUNX1, WT1.Conclusion①the patients with with t(16;21)(p11;q22)/FUSERG positive had unique clinical and laboratory features; they are hard to achieve CR in first chemotherapy, had high incidence of relapse within 2 years and a median of survival was only 8 months. Thus, the patients should be treated by other more powerful modality like stem cell transplantation in the first remission.②We established a new human acute myeloid leukemic cell line, JIH 4, with t(16;21)(p11;q22)/FUS-ERG fusion. It has clear biological characteristic and provided a powerful tool.