CLINICAL SIGNIFICANCE AND PROGNOSTIC VALUE OF SURVIVIN AND P53 IN CHILDREN WITH ACUTE LYMPHOBLASTIC LEUKEMIA

Background : Survivin is a member of the inhibitors of apoptosis (IAP) family , while P53 is a tumor suppressor protein which rapidly increases in response to cell stress , such as DNA damage. Both of Survivin and P53 are overexpressed in cancers including hematologic malignancy. Objective: To evaluate the level of survivin and P53 in children with acute lymphoblastic leukemia and to correlate it with clinical and hematological findings and response to treatment. Methods : The level of survivin was measured by ELISA and P53 expression was measured by flowcytometry in 37 children with acute lymphoblastic leukemia and 12 healthy children as a control group. The level of these two parameters was measured before and after treatment of patients and also correlated with clinical and hematological findings and response to treatment. Results : There was a highly significant difference in both serum survivin level and P53 expression between children with acute lymphoblastic leukemia at diagnosis compared to the control group , and the level has been significantly reduced after complete remission. Moreover , a significant positive correlation was found between serum survivin and LDH , WBCs count and percentage of blast cells in peripheral blood and bone marrow. Also , a significant positive correlation was found between survivin and P53 levels. Conclusion : The level of both survivin and P53 is significantly high in children with acute lymphoblastic leukemia and is positively correlated with poor prognostic factors , also survivin and P53 are positively correlated with each other.


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1. Complete Blood Count (CBC) using Sysmex KX 21. N. cell counter and examination of Leishman stained peripheral blood smear 2. Bone marrow aspiration for patients only, with examination of Leishman stained smears at diagnosis and after 28 days of induction of chemotherapy 3. Immunophenotyping on B.M. samples by flowcytometry for patients of acute leukemia at diagnosis using the following monoclonal antibodies: a. Markers of immature cells: HLA-DR and CD 34. b. Myeloid markers: CD 13 and CD 33. c. B-cell markers: CD 10, CD 19, CD 20 and CD 79b. d. T-cell markers: CD 2, CD 5 and CD 7. 4. Biochemical investigations using Cobas Integra 400 chemistry analyzer and including Liver function tests , serum LDH and serum creatinine.

Specific investigations:
Measurement of serum survivin levels by Enzyme Linked Immunosorbent Assay (ELISA) using Quantikine human survivin R & D System kit , it was done once for controls and twice for ALL patients ( at diagnosis and at complete remission).
Principle of the assay : Quantitative sandwich enzyme immune assay technique using a microplate pre-coated with monoclonal antibody specific for survivin. Standards and samples are pipetted into the wells and any survivin present is bound by the immobilized antibody. After washing of any unbound substances , an enzyme-linked polyclonal antibody specific for survivin is added to the wells. Any unbound antibody-enzyme reagent is then removed by washing followed by adding a substrate solution. A color will develop in proportion to the amount of survivin bound in the initial step.
Assay procedure: 1. Standards with concentrations of 2000 , 1000 , 500 , 250 , 125 , 62.5 and 31.25 pg/ml were added to the appropriate wells. 2. 100 ul of assay diluent were put into each well 3. 100 ul of controls were pipetted into appropriate wells 4. 100 ul of the samples were pipetted into appropriate wells 5. The plate was tapped gently for mixing , then sealed and incubated at room temperature on a plate shaker for 2 hours. 6. The wells were washed 5 times 7. 200 ul of survivin conjugate were pipetted into each well 8. Plate was sealed and incubated at room temperature on a plate shaker for 2 hours. 9. The wells were washed 5 times 10. 200 ul of substrate solution were pipetted into each well , then incubated at room temp. for 30 min. protected from light. 11. 50 ul of stop solution were pipetted into each well. 12. Optical density was measured within 30 min. by microplate reader at 450 nm Calculation of results: Standard curve was created by plotting the mean absorbance of each standard concentration on the y-axis against the survivin standards concentration on the x-axis. A curve was drawn through the points on the graph and the concentration of each sample was calculated from the curve.

Measurement of P 53 levels by flowcytometry:
It was done once for controls and twice for ALL patients ( at diagnosis and at complete remission).
Cells were incubated for 10 minutes at room temperature with 2 ml of mixture of 4 % paraformaldehyde and Becton Dickinson's FACS lysing solution. This procedure leads to lysis of RBCs and fixation and permeabilization of other 360 cells. Cells were centrifuged for 5 min. , the supernatant was discarded , and cells washed with 2 ml of 0.5 % Tween 20 in protein buffered saline (PBS) and centrifuged for 5 min. [16] P53 protein immunostaining : Fixed and permeabilized cells were incubated with 10 ml of anti-p53 Monoclonal Ab labeled with FITC (DO7 / DAKO, Carpintaria , CA) for 30 min. , followed by 2 washes with Tween 20/PBS , resuspended in 500 ul of 1 % formaldehyde / PBS and analyzed by FC.
Analysis was done with Fluorescence-activated cell analyzer (FACScan , San Jose , CA) with cell Quest software (Cell Quest™ software , Becton Dickinson Immunocytometry systems , San Jose , CA) Stained samples were analyzed through forward scatter (FSC) and side scatter (SSC) gated set around the blast cells population.
Expression was evaluated as cell percent (The number of stained cells minus the number of cells stained by irrelevant negative control).

Blast cells % in peripheral blood
The table shows that there is a highly significant difference regarding red blood cells (RBCs) , Hemoglobin , platelets and white blood cells (WBCs)      In our work , we studied the serum survivin level in children with acute lymphoblastic leukemia (ALL) at the time of diagnosis and also at complete remission in addition to a group of healthy children as a control group. Some hematological investigations related to acute leukemia were also done for our subjects.
There was a significant difference between control group and patients group at diagnosis regarding RBCs count , Hb level , platelets count , WBCs count , blast cells in peripheral blood and LDH level.
Also , we found a significant difference between patients at diagnosis and the same patients at complete remission regarding RBCs count , Hb level , platelets count , WBCs count , blast cells in peripheral blood , blast cells in bone marrow and LDH level. [19] also had found that level of LDH was significantly higher in patients with ALL at diagnosis than control group , while this level significantly decreased at remission , which agrees with our study.

Haviz and Mannan
Regarding serum survivin level , we found a highly significant difference between children with ALL at diagnosis compared to normal control group (p < 0.001). Also , survivin level has been significantly reduced after complete remission , so there was no significant difference between children with ALL at diagnosis and at complete remission. This agrees with Sadek et al., [20] and Raida et al., [21] 364 These findings also correlate with the study of Muhammad Al Makhbar and Almoutassem Billah Zetoune [22] who found that serum survivin levels were significantly greater in patients before therapy than after therapy.
Oto et al., [23] had studied serum survivin in patients with acute leukemia and concluded that it was high in those patients and had a bad prognostic effect while normal survivin level was associated with a good clinical outcome. Another study was done by Zhang et al., [24] and they stated that survivin level was significantly higher in children with acute lymphoblastic leukemia (ALL) and acute myeloid leukemia (AML) than in healthy children , and concluded that high survivin level has a role in modulation of apoptosis in patients with acute leukemia.
We also studied the correlations between serum survivin and hematological parameters and LDH level in our patients. A significant positive correlation was found with LDH , WBCs count and percentage of blast cells in peripheral blood and bone marrow (B.M.) , while a significant negative correlation was found with Hb , RBCs and platelets. This can be attributed to the anti-apoptotic effect of survivin that allows malignant (leukemic) cells to proliferate and invade B.M.
These correlations agree with the study of Paydas et al., [25] who found a positive correlation with LDH and with Nakayama and Kamihara [26] who found a positive correlation with WBCs count. Also ,our results agree with those of Kamal Elden et al., [27] who found a positive correlation of survivin expression with WBCs count and blast cells count in both peripheral blood and bone marrow.
So , according to our study , there is positive correlation of serum survivin with many unfavorable prognostic factors such as high percentage of blast cells , high LDH level , and increased WBCs count which can give important data about disease severity and progression. On the other hand , survivin level was decreased after complete remission so that it can be used for monitoring the response to therapy.
P53 is a very unstable protein due to its degradation by the proteasome after binding to its major negative regulator protein which is murine double minute 2 (MDM 2) [28]. When cells are exposed to stress such as DNA damage , P53 protein conformation will change and can escape the MDM 2 effect, accumulate and become an active transcription factor [29] and [30].
In this study , we found a highly significant increase in expression level of p53 in children with ALL at diagnosis compared to control group , while at complete remission the level decreased nearly to that of control group. This is in accordance with results of Park et al., [31] which showed that p53 was overexpressed in both acute lymphoblastic leukemia and acute myeloid leukemia patients by immunohistochemical technique.
Mohamed Abdel-Aziz [32] studied the serum level of p53 in patients with ALL and AML using ELISA technique and he found a significant increase of serum p53 in those patients compared to healthy control.
Another study that employed our technique , which is flowcytometry , for measurement of p53 was done by Konikova et al., [33] but it was done for AML cases only , and also it showed a significant high expression of p53 in AML patients compared to healthy people. Another study that agrees with our work and done also by flowcytometry was conducted by Raida et al., [21] and it included children with ALL. It revealed a highly significant elevation of p53 expression at diagnosis compared to healthy control while a significant decrease was detected at complete remission.
We also studied the correlation between the level of survivin and p53 , there was a significant positive correlation which is in agreement with the results of Raida et al., [21] and Hui et al., [34]. This correlation suggests that there is an interaction between p53 and survivin that can play a role in pathogenesis of ALL.

Conclusion:-
The level of both survivin and P53 is high in children with acute lymphoblastic leukemia and it drops nearly to normal after complete remission. Also , survivin and P53 are positively correlated with poor prognostic factors in those patients and are also positively correlated with each other. So , both of them can be used for monitoring the response to therapy in those patients.