Upregulation of SPINK2 in acute myeloid leukemia

Patient samples

In this retrospective study, 62 patients who were diagnosed at Istanbul University, Istanbul Medical Faculty, Pediatric Hematology Oncology and Department and Istanbul University-Cerrahpasa, Cerrahpasa Medical Faculty, Department of Internal Medicine, Division of Hematology were included. Blood or bone marrow samples were obtained for routine analysis with the informed consent from 17 pediatric (under 18 years) and 45 adult (18–80 years) patients with AML at diagnosis between 2013 and 2016. SPINK2 expression analysis was performed using leftover RNA samples of patients from routine fusion gene analysis. The control group consisted of three pediatric (under 18 years) and five adults (26–67 years old), who were bone marrow transplantation donors. Apart from healthy control, nine individuals without leukemia who underwent bone marrow biopsy due to the pre-diagnosis of leukemia were also used as another group of controls. The approval of Istanbul University Istanbul Medical Faculty Clinical Research Ethics Committee (reference number: 537, 24/05/2017) was obtained for this study. This study was conducted in accordance with the 1964 Declaration of Helsinki and its subsequent amendments. The demographic characteristics of the patients are shown in Table 1.

Cell lines

HL60 (acute promyelocytic leukemia), NB4 (human promyelocytic leukemia), K562 (chronic myeloid leukemia), Jurkat (acute T-cell leukemia) and NALM6 (acute lymphoblastic leukemia) cell lines were cultured in RPMI medium containing 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. MCF7 (epithelial cell obtained from mammary gland metastatic area), HeLa (cervical epithelial cell with adenocarcinoma), HUVEC (endothelial cell derived from the human umbilical cord), hFOB (human osteoblasts), 293T (human embryonic kidney epithelial cell), U87 (human brain epithelial cell line) were cultured in the DMEM medium, including 10% FBS, 2 mM L-glutamine, 100 units/mL penicillin and 100 μg/mL streptomycin. They were reproduced in a 5% CO₂ incubator at 37 °C.

Determination of SPINK2 mRNA expression in patients with AML and cell lines using qRT-PCR

RNA samples were extracted from cells using PureLink, RNA Mini Kit (Invitrogen, Carlsbad, CA, USA) following the protocol of the kit. Transcriptor High Fidelity cDNA Synthesis Kit (Roche Diagnostic, Mannheim, Germany) was used for RT-PCR according to the manufacturer’s instructions. cDNA was synthesized from 500 ng total RNA in 20 µL reaction volume.

A real-time quantitative PCR device (LightCycler, Roche Diagnostic, Mannheim, Germany) was used for quantitation. TATA binding protein (TBP) housekeeping gene was used as a control gene. Primer sequences and PCR product sizes are given in Table 2. SYBR Green master kit (Roche Diagnostic, Mannheim, Germany) and 1 µL cDNA sample were used for the 20 µL PCR reaction. The PCR conditions were as follows 60 s pre-denaturation at 95 °C, 10 s at 95 °C, 20 s at 68 °C and 10 s at 72 °C (45 cycles). After amplification, the program was applied for melting curve analysis for 0 s at 95 °C, 10 s at 65 °C and 0 s at 95 °C. The differences in SPINK2 expression in patients were determined using ∆∆Ct method according to the healthy control sample [15]. mRNA expression levels were indicated as arbitrary units (AU).

Determination of SPINK2 protein level using ELISA

The cultured cells were seeded with two million cells in 4 mL medium containing 0.2% FBS suitable for the cell type. After 48 h, cells were lysed in 200 µL of extraction solution (100 mM Tris, pH 7.4, 150 mM NaCl, 1 mM EGTA, 1 mM EDTA, 1% Triton X-100, 0.5% Sodium deoxycholate). Total protein concentration in cells was determined using the bicinchoninic acid assay (Pierce BCA Protein Assay kit, Thermo Fisher Scientific, Waltham, MA, USA). The Sandwich ELISA was performed according to the human serine protease inhibitor Kazal-type 2 (SPINK2) ELISA Kit (MyBioSource, San Diego, CA, USA) protocol. Samples were measured using the ELISA plate reader at 450 nm. Intracellular SPINK2 protein levels were normalized to total protein levels. All samples, standards and controls were run in triplicate. The mean value of triplicate samples was calculated.

Detection of fusion genes by RT-PCR analysis

AML1-ETO associated with t(8;21), CBFB-MYH11 with inv(16) and PML-RARA with t(15;17) fusion gene transcripts were detected by reverse transcriptase polymerase chain reaction (RT-PCR) [16].

Statistical analysis

The data were analyzed using the SPSS Statistics Program Version 20.0 (IBM SPSS Statistics for Windows, Version 20.0. Armonk, NY: IBM Corp.) and GraphPad Prism 7.0. Kolmogorov-Smirnov and Shapiro-Wilk tests was used to determine normality. Independent sample T-test for parametric data and Kruskal-Wallis and Mann-Whitney U tests were used for non-parametric data. To investigate the correlation, Spearman’s tests were used. Receiver operating characteristic curve (ROC) and the area under curve (AUC) were conducted to assess the value of SPINK2 expression for discriminating patients with AML from controls. Overall survival (OS) was defined as time from disease diagnosis to death from any cause or censoring for patients alive at their last known date of contact. The Kaplan-Meier analysis and log rank test were used to determine patients’ survival rates. p<0.05 was considered statistically significant for all statistical analyses.

SPINK2 expression in cell lines

qRT-PCR was used to determine the level of SPINK2 mRNA in 11 cell lines (Supplementary Figure 1). We were found that the expression of SPINK2 mRNA was higher in AML cell lines (median: 10.5 AU, range: 8.72–12,32 AU) than non-AML cell lines (median: 0.9 AU, range: 0.41–3.41 AU) using Mann-Whitney U test (p=0.036). SPINK2 mRNA expression in leukemia cell lines (median: 3.4 AU, range: 2.39–12.32 AU) also significantly increased compared to non-leukemia cell lines (median: 0.7 AU, range: 0.41–1.32 AU) (p=0.006).

SPINK2 protein levels found in the cell lines and proteins secreted into the medium were determined by ELISA method. The intracellular and extracellular SPINK2 protein levels are given in Supplementary Figure 1. The intracellular and extracellular SPINK2 expression in AML cell lines (median: 0.074 ng/mg, range: 0.95–1.04 ng/mg and median: 3.85 ng/mg, range: 0.05–0.30 ng/mg, respectively) also significantly increased compared to non-AML cell lines (median: 0.056 ng/mL, range:0.05–0.079 ng/mL and median: 0.004 ng/mL, range: 0–0.02 ng/mL, respectively) (p=0.036 and p=0.023, respectively).

SPINK2 mRNA was correlated with intracellular and extracellular protein (p=0.0002, R=0.935 and p=0.046, R=0.610 respectively). There was also a statistically significant correlation between the intracellular and secreted protein levels (p=0.002, R=0.817).

SPINK2 expression and its prognostic significance in patients

SPINK2 mRNA levels in patients with AML (median: 1.62 AU, range: 0.0032–28.67 AU) were significantly higher than in controls (median: 0.058 AU, range: 0.00017–1.51 AU), which showed that SPINK2 expression was significantly up-regulated in AML (p=0.004) (Figure 1). We also used nine samples from individuals without leukemia who underwent bone marrow biopsy due to the pre-diagnosis of leukemia as another group of control apart from healthy control samples. The median value of SPINK2 mRNA expression was 0.28 AU (range: 0.035–1.15 AU). We observed that SPINK2 was significantly increased in AML, even compared with nine samples without leukemia (p=0.022).

Figure 1:

The median expression level of SPINK2 mRNA in healthy controls and patients with AML. SPINK2 mRNA levels were determined using cDNA derived from 25 ng total RNA. The relative mRNA expression levels were calculated using the comparative CT method. The long horizontal lines above the dots represent the median. The short horizontal lines above the dots represent the median interquartile range. AML, acute myeloid leukemia.

Additionally, ROC curves were generated to evaluate the diagnostic value of SPINK2 expression in AML. ROC analysis revealed that SPINK2 is a well diagnostic marker with the AUC value of 0.82 [95% confidence interval (CI): 0.685–0.946] (p=0.004). The result is shown in Figure 2.

Figure 2:

ROC curve of SPINK2 mRNA expression in AML. AML, acute myeloid leukemia.

The comparisons of SPINK2 expression according to the clinical and laboratory characteristics of patients with AML are given in Table 3. There was no significant relationship between SPINK2 expression and age, gender, white blood cell count and French American British (FAB) groups in patients (Table 3).

SPINK2 expression was analysed in AML patients with t(15;17), t(8;21), inv(16) mutations using the Mann Whitney U test (Table 3). A statistically significant difference was found between t(8;21)-positive and negative patients. SPINK2 expression was significantly lower in t(8;21)-positive than in negative patients with AML (p=0.00006).

We categorized the patients (n=50) into two groups (SPINK2 ^high and SPINK2 ^low) based on the median expression level of SPINK2 mRNA (1.62 AU) to evaluate the prognostic value of SPINK2 expression. According to Kaplan-Meier analysis, the 7-year overall survival rates were 27.9 ± 8.4%, for SPINK2^high and 40.6 ± 11.6% for SPINK2 ^low patients. There was no significant difference in OS between SPINK2 ^high and SPINK2 ^low patients (log-rank p= 0.40) (Figure 3). Median survival times were 15 (range: 1–92) months for SPINK2 ^high and 20.5 (range: 3–89) months for SPINK2 ^low patients.

Figure 3:

Impact of the SPINK2 expression on the overall survival rate of patients with AML. Overall survival was evaluated by the Kaplan-Meier curve. The cut-off value was determined at the median expression level (1.62 AU) to define the groups. AML, acute myeloid leukemia.