Abstract 4910

The Kindlin family of intracellular proteins has recently emerged as key regulators of cellular functions and cell-matrix interactions. They comprise of three evolutionarily conserved members, kindlin-1, kindlin-2 and kindlin-3, they share considerable sequence and structural similarities. A few of study revealed that Kindlin-2 influences solid tumor cell invasion and resistance. With regard to AML, the influence of Kindlins is still unknown. To evaluate the clinical significance of Kindlin-2 in acute myeloid leukemia (AML), we investigated the expression of Kindlin-2, kindling-3 in AML cells.

1. Materials and methods

K562, KG-1a, HL60, U937, Jurkat cell lines were cultured in RPMI 1640 medium, supplemented with 10% fetal bovine serum (FBS, GIBCO) at 37°C in a humidified atmosphere of 5% CO2. Bone marrow (BM) samples were obtained from 88 patients with de novo AML from Blood Diseases Hospital, Chinese Academy of Medical Sciences & Peking Union Medical College (CAMS & PUMC). Samples of 9 normal donors and ITP were used as the control group.

Bone marrow mononuclear cells (BMMCs) were prepared by Ficoll-Hypaque density gradient centrifugation. Expressions of Kindlin-2, Kindlin-3 were detected by RQ-PCR. The following primers for real-time PCR were used: (a) Kindlin-2 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (b) Kindlin-2 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (c) Kindlin-3 sense primer, 5'-CCGCTCGAGCTATGCGTATCCCCGTAG-3'; (d) Kindlin-3 antisense primer, 5'-CGACGCGTCTAGCGAGGGGTTGTC-3'; (e) GAPDH sense primer, 5'-GAAGGTGAAGGTCGGAGTC-3'; (f) GAPDH antisense primer, 5'-GAAGATGGTGATGGGATTTC-3'. Analysis was performed using ABI 7500 Sequence Detection software (Applied Biosystems). The expression of Kindlin-2 and Kindlin-3 were showed as RQ value calculated through ΔΔCt method [ΔΔCt = (CtKindlin □ CtGAPDH)sample □ (CtKindlin □ CtGAPDH)calibrator]. The ΔCt (CtKindlin □ CtGAPDH) of K562 was defined as calibrator, and the RQ of calibrator was 1.000. Relationships between Kindlin-2, Kindlin-3 and the patients' clinical data were analyzed.

2. Results
Expression of Kindlins in newly diagnosis AML
Table 1.

Characteristics of the AML patients and expression of Kindlins

CharacteristicNo. of Patients (%)Kindlin-2 (RQ-In)Kindlin-3 (RQ-In)
Sex    
Male 46(52.3%) 0.307±1.659 0.066±0.738 
Female 42(47.7%) 0.005±1.676 0.117±0.942 
p-value  0.398 0.776 
WBC    
<30×109/L 22(25.3%) 0.409±1.534 −0.013±0.753 
≥30×109/L 65(74.7%) -0.547±1.893 0.394±1.021 
p-value  0.020 0.049 
WHO type    
AML with t(8;21)(q22;q22) 16(18.2%) 0.842±1.730 −0.207±0.811 
AML with t(15;17)(q22;q12) 14(15.9%)   
Acute myelomonocytic leukaemia 5(5.7%) −0.240±1.473 0.243±0.790 
Acute monoblastic and monocytic leukaemia 31(35.2%)   
AML without maturation 3(3.4%) −0.148±1.630 0.267±0.869 
AML with maturation 13(14.8%)   
Acute erythroid leukaemia 6(6.8%)   
p-value  0.016 0.046 
NCCN risk status(APL excluded)    
Better-risk 20(27.0%) 0.446±1.888 0.057±1.056 
Intermediate-risk 41(55.4%) 0.027±1.469 0.17±0.741 
Poor-risk 13(17.6%) -0.376±1.500 0.347±0.794 
p-value  0.347 0.630 
Induction chemotherapy 39   
Pretherapy 39 0.216±1.602 −0.114±0.628 
Postremission 39 1.555±1.102 0.372±0.633 
p-value  <0.001 0.004 
CharacteristicNo. of Patients (%)Kindlin-2 (RQ-In)Kindlin-3 (RQ-In)
Sex    
Male 46(52.3%) 0.307±1.659 0.066±0.738 
Female 42(47.7%) 0.005±1.676 0.117±0.942 
p-value  0.398 0.776 
WBC    
<30×109/L 22(25.3%) 0.409±1.534 −0.013±0.753 
≥30×109/L 65(74.7%) -0.547±1.893 0.394±1.021 
p-value  0.020 0.049 
WHO type    
AML with t(8;21)(q22;q22) 16(18.2%) 0.842±1.730 −0.207±0.811 
AML with t(15;17)(q22;q12) 14(15.9%)   
Acute myelomonocytic leukaemia 5(5.7%) −0.240±1.473 0.243±0.790 
Acute monoblastic and monocytic leukaemia 31(35.2%)   
AML without maturation 3(3.4%) −0.148±1.630 0.267±0.869 
AML with maturation 13(14.8%)   
Acute erythroid leukaemia 6(6.8%)   
p-value  0.016 0.046 
NCCN risk status(APL excluded)    
Better-risk 20(27.0%) 0.446±1.888 0.057±1.056 
Intermediate-risk 41(55.4%) 0.027±1.469 0.17±0.741 
Poor-risk 13(17.6%) -0.376±1.500 0.347±0.794 
p-value  0.347 0.630 
Induction chemotherapy 39   
Pretherapy 39 0.216±1.602 −0.114±0.628 
Postremission 39 1.555±1.102 0.372±0.633 
p-value  <0.001 0.004 

The level of Kindlin-2 in AML (0.163±1.665) was significantly lower than that in non-AML (1.683±1.395) controls (p=0.010). No significant difference was found between the AML and controls in levels of Kindlin-3 (p=0.216). Out of the 79 patients who accepted treatment, 61 patients achieved complete remission (CR) and 18 patients were NR. Patients with higher expression of Kindlin-2 had a higher CR rate (86.8% vs 68.3%) (p=0.050). Expression of kindling 3 was unrelated to CR rate. Both of kindling-2 and kindling-3 increased after CR. This finding implicates Kindlin-2 as a potential prognostic factor of AML.

Disclosures:

No relevant conflicts of interest to declare.

Author notes

*

Asterisk with author names denotes non-ASH members.