Simultaneous Analysis Of Telomere Length And Cell Surface Antigen In Leukemia By Multicolor FLOW-FISH

Background and objective:Telomeres are specialized structures of the ends of eukaryon linear chromosomes, consisting of repetitive non-encoding(TTAGGG)n DNA sequences.Its function as an end-protector of chromosomes prevents the chromosome from end-to-end fusion, recombination and degradation.Telomerase acts as reverse transcriptase in the elongation of telomeres,which prevents the loss of telomeres due to the end replication problems.During the last decade telomere length and telomerase have been discovered as new biological markers and potential new targets of cancer.There are also many reseaches about telomere length and telomerase activity in malignant hematopoietic disorders.Most studies show that acute leukemia have reduced telomeres at diagnosis and restored telomeres are found with clinical remissions. Patients with short telomeres seem less likely to achieve a complete remission.The telomere length of patients in CML-CP is normal or reduced,however in AP and BC a pronounced reduction is observed.Imatinib treatment might lead to a normalization of shortened telomeres.It is possible that telomere loss is responsible for imatinib resistance and CML progression.A correlation between progressive telomere shortening and CLL progression is also demonstrated.Patients with short telomeres seem have shorter median overall survival(OS).In MDS,telomere shortening is evident in about 40%of patients during the disease progression,and patients with short telomeres at diagnosis seem more likely to transform into acute leukemia.Thus, the measurement of telomere length is of great clinical significance.Meanwhile,the modifications in methods of telomere length measurement have drawn clinical workers' attention.A FLOW-FISH method,i.e.quantitative fluorescence in situ hybridization(Q-FISH) in combination with flow cytometry (FCM),has been developed recently.In FLOW-FISH a fluorescein isothiocyanate (FITC)-labeled telomere-specific peptide nucleic acid(PNA) probe is hybridized in a quantitative way to telomere repeats,followed by telomere fluorescence measurements on individual cells by flow cytometry.Compared with Southern blot and Q-FISH,FLOW-FISH offers the advantages of looking at telomere fluorescence in different subpopulations of cells in the same sample and many samples with relatively low cell counts(10⁵) or with non-dividing cells can be processed and less time-consuming.Most overseas studies detect telomere length by FLOW-FISH without combined analysis of cell immunophenotype,thus the results only represent the mean telomere length of the whole cells and the little leukemic cells with short telomeres might not be discriminated from the most normal cells,which limit its significance in minimal residual disease(MRD) detection.In addition,during leukemic progression the biological characteristics of leukemic cells might change such as the occurrence of lineage transformation from acute myeloid leukemia to acute lymphocytic leukemia.The single detection of telomere length could not monitor such clonal evolution as early as possible.On the other hand,although the single analysis of cell immunophenotype is of great meaning in the diagnosis of de novo leukemias,it is difficult to distinguish leukemic cells with the same differentiation antigen expression from their normal counterparts for monitoring MRD.MRD studies is significant for monitoring leukemia,however,only a small part cases have specific chromosomal aberrations for cytogenetical or molecular studies of MRD.There are not good markers for MRD studies in most patients,thus it is necessary and urgent to find one generally applied method.FCM holds one great advantage for immunological studies of MRD because of its wide availability, otherwise it has specific limitation as stated above,that is its difficulty to distinguish the abnormal and normal clone.Therefore,the combination of one marker which could distinguish the abnormal and normal clone might offset the limitation and offer much help for monitoring leukemia.This research aimed at establishing the reliable FLOW-FISH method of detecting telomere length in our laboratory and observing the dynamics of telomere length in different leukemia in order to obtain further insight into the related pathogenesis of leukemia.In addition,we analyzed telomere length and cell immunophenotype simultaneously in the same sample by FLOW-FISH in order to combine the two markers to found one generally applied method for monitoring disease conditions or predicting clinical outcome in leukemia.Methods:1.HL60,Raji,Molt4 cells were cultivated.Each step and the conditions of the FLOW-FISH procedure were optimized,standardized and validated,then the related operation rules and analysis strategy were provided.The relative telomere length (RTL) of the leukemic sample cells compared to the control cells(Molt4) were calculated in the following way: 2.Bone marrow samples were derived from 34 leukemia patients,who were finally diagnosed by the means of cell morphology,immunology and cytogenetics.20 normal peripheral blood samples from healthy individuals were set as control group.3.The bone marrow or peripheral blood mononuclear cell(MNC) were collected and used for FLOW-FISH.14 acute leukemia patients undergone simultaneous analysis of telomere length and cell differentiation antigen before and after treatment.The rest 20 patients and the 20 normal controls undergone the single detection of telomere length.Meanwhile,the RQ-PCR measurements in the same samples were performed to test the correlation of the two methods.We compared telomere length of the patients and normal controls and analyzed the relationship between telomere length and clinical character,therapeutic effect and prognosis preliminarily.As for those tracing patients,we observed the changes of telomere length combined with immunophenotype in different courses of disease.4.Statistical analysis:The Mann-Whitney U test was used to compare two independent groups of data and the Wilcoxon signed rank test for two related groups. The Kruskal-Wallis test was used to compare more than three groups of data. Associations among variables were assessed using the Spearman rank correlation analysis.The level of signifcance was considered as P＜0.05 and the P values were two-tailed.Results:1.By using various cell lines the reliable method of detecting telomere length by FLOW-FISH was established,meanwhile,simultaneous analysis of telomere length and cell differentiation antigen by multicolor FLOW-FISH was also successfully established.2.The median of RTL in 24 acute leukemia patients was 0.240,2 CLL was 0.235,3 CML-BP was 0.249,5 CML-CP was 0.435,while that in 20 normal controls was 0.408.There was significant difference among the groups(x²=18.403,P= 0.001).The telomere length was shorter in patients with acute leukemia,CLL, CML-BP than in normal controls,otherwise,the CML-CP cases had no difference with the controls.14 acute leukemia patients had the significantly longer telomere with clinical remissions than at impetus stage(Z=-3.233,P=0.01),and no significant difference was found between the 12 cases with clinical remissions and the normal controls(U=114.0,P=0.363).A high degree of correlation between the RTL obtained from FLOW-FISH and telomere length estimation by RQ-PCR existed(r_s=0.860,P=0.000).3.There was a significant negative correlation between telomere length and age in 20 normal controls(r_s=-0.671,P=0.001),and the female had longer telomere than the male(U=22.0,P=0.037).However no statistic difference between telomere length and age or gender existed in 34 de novo leukemia patients.The telomere length of 24 acute leukemia cases had a negative correlation with peripheral white cells counts(r_s=-0.597,P=0.002).But no significant correlation was found with hemoglobin and platelet counts.Within 23 follow-up acute leukemia patients,the CR rate in cases with telomere length above the median was 81.82%, and 58.23%in the rest.Among 5 CML-CP patients who received imatinib treatment, 2 cases with the shorter telomere(RTL 0.254～0.232) occurred transformation to BP in less than 6 months,the other 3 cases(RTL 0.435～0.598) obtained CHR within 3 months,CCyR at 6 months and no transformation to BP was observed in our follow-up of 10 months.4.14 acute leukemia patients who undergone simultaneous analysis of telomere length and immunophenotype show the coincidence with the convention immunology in cell differentiation antigen expression.Telomere length combined with cell differentiation antigen of 5 cases were dynamically detected at different courses of disease.It showed that telomere length of 2 cases with continued CR restored after CR and remained at normal level during remission,and no specific antigen expressed highly.However,telomere of 3 relapsed cases were significantly shortened at impetus stage,prolonged after CR and shortened again after relapse.In 2 relapsed cases,the telomere of related antigen positive cells shortened before the whole cells,and ahead of cell morphology change.Conclusions:1.Two methods of detecting telomere length by FLOW-FISH and RQ-PCR (2^{-ΔΔCT}) were established.2.Simultaneous analysis of telomere length and cell differentiation antigen by multicolor FLOW-FISH was established.3.The telomere length was significantly shorter in 34 de novo leukemia patients than in normal controls except the CML-CP cases.Restored telomeres were found with clinical remissions in 14 acute leukemia cases.The two methods of FLOW-FISH and RQ-PCR generated the same results and showed notable correlated.4.There was a significant negative correlation between telomere length and age in normal controls,and the female had longer telomere than the male.However no statistic difference between telomere length and age or gender existed in de novo leukemia patients.The telomere length of 24 acute leukemia cases had a negative correlation with peripheral white cells counts.It seemed the shorter telomere mean the lower CR rates in 23 follow-up cases with acute leukemia and the shorter duration of CP before onset of BP in 5 cases with CML. 5.Simultaneous analysis of telomere length and cell differentiation antigen by multicolor FLOW-FISH made for overall monitor for leukemia and might be a new generally applied method for monitoring MRD in leukemia.