Simultaneous Analysis of Telomere Lengthes and Cell Surface Antigen in Leukemia by Multicolor Flow-FISH

During the last decade telomere length (TL) and telomerase have been discovered as new biological markers and potential new targets of cancer including malignant hematopoietic disorders. A FLOW-FISH method, i.e. quantitative fluorescence in situ hybridization (Q-FISH) in combination with flow cytometry (FCM), has been developed recently. Most overseas studies detect TL by FLOW-FISH without combined analysis of cell immunophenotype. We analyzed TL 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. The relative TL (RTL) of the leukemic sample cells compared to the control cells(Molt4).

2. BM samples were derived from 34 leukemia patients. 20 normal PB samples from healthy individuals were set as control group.

3. The BM or PB MNC were collected and used for FLOW-FISH. Meanwhile, the RQ-PCR measurements in the same samples were performed to test the correlation of the two methods.

Results:

1. Using various cell lines, multicolor FLOW-FISH was also successfully established.

2. The median of RTL in 24 AL 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.(P=0.001). The TL was shorter in patients with AL, CLL, CML-BP than in normal controls, otherwise, the CML-CP cases had no difference with the controls. 14 AL patients had the significantly longer telomere with clinical remissions than at impetus stage (P=0.01), and no significant difference was found between the 12 cases with clinical remissions and the normal controls(P=0.363). A high degree of correlation between the RTL obtained from FLOW-FISH and TL estimation by RQ-PCR existed (P=0.000).

3. There was a significant negative correlation between TL and age in 20 normal controls(P=0.001), and the female had longer telomere than the male(P=0.037). However no statistic difference between TL and age or gender existed in 34 de novo leukemia patients. The TL of 24 AL cases had a negative correlation with peripheral white cells counts(P=0.002). Within 23 follow-up AL patients, the CR rate in cases with TL 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 occurred transformation to BP in less than 6 months, the other 3 cases 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 AL patients who undergone simultaneous analysis of TL and immunophenotype show the coincidence with the convention immunology in cell differentiation antigen expression. TL combined with cell differentiation antigen of 5 cases were dynamically detected at different courses of disease. It showed that TL 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. The TL 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 AL cases. The two methods of FLOW-FISH and RQ-PCR generated the same results and showed notable correlated.

2. There was a significant negative correlation between TL and age in normal controls, and the female had longer telomere than the male. However no statistic difference between TL and age or gender existed in de novo leukemia patients. The TL of 24 AL 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 AL and the shorter duration of CP before onset of BP in 5 cases with CML.

3. Simultaneous analysis of TL 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.