Abstract
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.
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.
Characteristics of the AML patients and expression of Kindlins
| Characteristic . | No. 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 |
| Characteristic . | No. 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.
No relevant conflicts of interest to declare.
Author notes
Asterisk with author names denotes non-ASH members.
