MDR and Cyclin A2

Slide 1: 

The Correlation of Cyclin A2 with Drug Resistance in Adult Acute Non Lymphoblastic Leukemia

THE CELL CYCLE : 

THE CELL CYCLE G1 = growth and preparation for replication S = synthesis of DNA . G2 = preparation for M = mitosis

Cyclin and Cyclin depended Kinas : 

Cyclin and Cyclin depended Kinas The passage of a cell through the cell cycle is controlled by proteins in the cytoplasm called cyclins 1- G1 cyclin (cyclin D) 2- S-phase cyclins (cyclins E and A) 3- mitotic cyclins (cyclins B and A) Their levels in the cell rise and fall with the stages of the cell cycle. - Cyclin-dependent kinases (Cdks) 1- G1 Cdk (Cdk4) 2- S-phase Cdk ((Cdk2) 3- M-phase Cdk (Cdk1) Their levels in the cell remain fairly stable, but each must bind the appropriate cyclin (whose levels fluctuate) in order to be activated

Cell Cycle under Control of Cyclins : 

Cell Cycle under Control of Cyclins Cyclin D control G1 phase . Cyclin A & E control S phase Cyclin B control G2 and Mitosis

Function of Cyclin A2 : 

Function of Cyclin A2 Cyclin A2 level increase during S phase and then bound to Cdk2 and enter the nucleus to prepare the cell to duplicate its DNA ( and its centrosomes). Cyclin A2 achieve its maximum peaks during S and G2 phases.

History of MDR-1 : 

History of MDR-1 In 1970 , MDR 1 in mammalian cells was first described , When resistance to alkaloids and various toxic agent was associated with an energy-dependent cellular transmembrane drug efflux. Later it was found that this phenomena is associated with emergence of a cell membrane–bound glycoprotein designated P-gp. In 1987, The gene (MDR1) encoding for P-gp was assigned to the long arm of chromosome 7 (7q21-31) . In 1990 , complementary DNA (cDNA) sequence and genomic organization of the MDR1 respectively were become known . The development of monoclonal antibodies against P-gp allowed for assessment of the significance of P-gp over expression in clinical specimens.

MDR -1 (P-gp glycoprotein) : 

MDR -1 (P-gp glycoprotein)

Slide 8: 

Mechanisms of Drug Resistance

Slide 9: 

Our aim is trying to know if there is a correlation between Cyclin A2 and MDR-1 in acute non lymphoblastic leukemia in adults and what type of this correlation ?!

Patients and Methods : 

Patients and Methods

Slide 11: 

52 newly diagnosed with AML The diagnosis of AML were based on FAB classification. The studied group was subjected to through history, physical examination, complete blood picture, bone marrow examination , immunophenotyping to confirm diagnosis of AML ,and cytogenetics when needed . Other laboratory tests were also done including, liver and kidney function tests. Radiological examination including chest x ray, abdominal ultrasound and/or CT scan were done whenever needed for proper clinical diagnosis. Patients were followed up for 20 months to see their response to the treatment

Slide 12: 

Both Cyclin A 2 and MDR-1 were measured in the studied group and 10 normal control by : - RT-PCR ( gene activity ). - Flowcytometry ( gene products ).

Result : 

Result

Clinical data of the study group : 

Clinical data of the study group

Laboratory Data of the study group : 

Laboratory Data of the study group

Types o leukemia : 

Types o leukemia 17 patients M0 and M1 (33%) 22 patients M2 (42%) 9 patients M3 ( 17%) 4 patients M4 (8%)

Cyclin A2 protein by Flow Cytometry : 

Cyclin A2 protein by Flow Cytometry

Cyclin A2 by PCR : 

Cyclin A2 by PCR

Cyclin A2 by Flow and RT-PCR : 

Cyclin A2 by Flow and RT-PCR

MDR-1 by Flow Cytometry : 

MDR-1 by Flow Cytometry

MDR-1 by RT-PCR : 

MDR-1 by RT-PCR By RT-PCR 24 patients out of 52 (45%) were positive and 28 patients were negative

MDR-1 in the study group by Flow and RT-PCR : 

MDR-1 in the study group by Flow and RT-PCR

Correlation Between Cyclin A2 and MDR-1 : 

Correlation Between Cyclin A2 and MDR-1 On studying the expression of Cyclin A2 and MDR-1 gene expression by RT-PCR, There was a significant inverse relationship between them (? = 0.005). Higher values of Cyclin A2 is present in MDR-1 negative patients while lower value were detected in MDR-1 positive patients . The mean value ± 2SD of cyclin A2 by flow cytometry was 0.53 ± 0.63 in cases showing positive MDR-1 expression, while it was 1.56 ± 2.48 in cases showing negative MDR-1 expression. With no statistically significant difference P =0.141

Correlation between Cyclin A2 & MDR-1by RT-PCR & Flow Cytometry : 

Correlation between Cyclin A2 & MDR-1by RT-PCR & Flow Cytometry

Correlation between Cyclin A2 and MDR1by RT-PCR Higher value of Cyclin A2 is present in MDR-1 negative patients : 

Correlation between Cyclin A2 and MDR1by RT-PCR Higher value of Cyclin A2 is present in MDR-1 negative patients

Study group and responseto treatment : 

Study group and responseto treatment Thirty patients (59%) showed CR and PR while twenty one (41%) were, Refractory or resistant to treatment, one case is missing.

Relationship between Cyclin A2 expression and Response to treatment : 

Relationship between Cyclin A2 expression and Response to treatment By RT-PCR, the rate of CR and PR was significantly higher in the group of positive expression of Cyclin A2 compared to that of negative expression (p=0.02). By flow cytometry, the rate of Cyclin A2 expression was higher in the group of CR and PR 23/30 (77%) patients as compared to the resistant group 13/21 (62%), but the difference was not statistically significant (p=0.255)

Cyclin A2 and Response : 

Cyclin A2 and Response By RT-PCR, the rate of CR and PR was significantly higher in the group of positive expression of Cyclin A2 compared to that of negative expression (p=0.02). In CR & PR group 21/30( 70%) patients showed high A2 level In Ref. group only 8/21(38%) patients showed high A2 level

MDR -1 and Response by Flow cytometry : 

MDR -1 and Response by Flow cytometry The rate of CR and PR in the group with negative MDR-1 expression was significantly higher than the group with positive MDR-1 expression. This result was detected by flow cytometry and RT-PCR (p=0.005 , 0.004 respectively)

MDR-1 and response by RT-PCR : 

MDR-1 and response by RT-PCR The rate of CR&PR in Negative MDR-1 patients is significantly higher than the group of positive MDR-1 , P = 0.004

Overall Survival in relation to Cyclin A2 & MDR-1 : 

Overall Survival in relation to Cyclin A2 & MDR-1 SE = Standard error * = Significant

Cyclin A2 and Survival : 

Cyclin A2 and Survival The Overall Survival (OS) in the group with positive Cyclin A2 Expression (37/52) was significantly higher than in the group of negative Cyclin A2 expression (15/52), p = 0.03. This significant relationship was detected only by Flow cytometry. By RT-PCR technique, The OS was also higher in Cyclin A2 positive cases compared to negative cases , however It did not reach a statistically significant level p=0.48

MDR-1 and Survival : 

MDR-1 and Survival The OS in the group with negative MDR1 expression was, significantly higher than that in the group of positive MDR1 expression (24/52) p= 0.04. This relationship is detected only by RT-PCR however, by flow cytometry it was non significant p = 0.95

Disease free survival (DFS) in relation to Cyclin A2 and MDR1 : 

Disease free survival (DFS) in relation to Cyclin A2 and MDR1 Cyclin A2 the disease free survival ( DFS) in the group with positive Cyclin A2 expression (23/30) was higher compared to the group of negative CyclinA2 expression (7/30) , however the difference was statistically non-significant either by RT-PCR or by flow cytometry ( p=0.14 , 0.34 respectively ) MDR The DFS in the group with negative MDR1 expression (32/52) is significantly higher than in the group of positive MDR1 expression (7/52), p=0.03. This relationship is detected only by flow cytometry, By RT-PCR technique, it was non significant, p=0.46

Conclusion : 

Conclusion It is concluded that lower expression of cyclin A2 might be an unfavorable prognostic factor for patients with AML . This study also confirm the clinical relevance of MDR-1 and the measurement of cyclin A2 and MDR-1 would help predict prognosis and treatment result of AML patient .   We hope that this study to be of benefit to guide chemotherapy and MDR-1 modulators .

Thank You : 

Thank You