Methylation-independent CRIP1 expression is a potential biomarker affecting prognosis in cytogenetically normal acute myeloid leukemia

Abstract

Abnormal expression of CRIP1 has been identified in numerous solid tumors. However, CRIP1 expression and its regulation are little known in acute myeloid leukemia (AML). The purpose of this study was to evaluate the expression and regulation of CRIP1 and the clinical implications of CRIP1 aberration in AML. Real-time quantitative PCR was carried out to detect the level of CRIP1 expression in 138 AML patients and 38 controls. CRIP1 methylation was detected by methylation-specific PCR and bisulfite sequencing PCR. Five public available AML datasets from The Cancer Genome Atlas (TCGA) and Gene Expression Omnibus (GEO) were further analyzed. The level of CRIP1 expression was up-regulated in AML patients compared with controls (P = 0.045). CRIP1 ^high patients had a significantly lower complete remission (CR) rate than CRIP1 ^low patients (P = 0.020). CRIP1 ^high group had a shorter overall survival (OS) and leukemia-free survival (LFS) than CRIP1 ^low group in cytogenetically normal AML (CN-AML) patients (P = 0.007 and 0.012, respectively). Multivariate analysis further confirmed that high CRIP1 expression was an independent risk factor for LFS in CN-AML patients (P = 0.005). However, we found that CRIP1 expression was not associated with the status of its promoter, which was nearly fully unmethylated both in controls and AML patients. Furthermore, our results were validated using the published GEO datasets and TCGA datasets. Our findings suggest that high CRIP1 expression is independently related with unfavorable prognosis in CN-AML.

Keywords: AML; CRIP1; biomarker; methylation; prognosis.

Figure 1

Identification of potential oncogenes in AML. A, B: Heatmaps showing 68 up-regulated genes in controls versus AMLs, from GSE24006 and GSE63270, respectively. Log2 fold changes of gene expression (log2 FC expression) are displayed as bar graphs on the right. C: Venn diagram showing the overlap of four gene sets including: 1031 up-regulated genes (FDR < 0.05, log2 FC > 2) in controls versus all AML patients and 348 up-regulated genes in LSCs versus HSCs in GSE24006 datasets (data 1), 535 up-regulated genes in controls versus all AML patients and 357 up-regulated genes in LSCs versus HSCs in GSE63270 datasets (data 2). The overlapping region (68) represents the finally screened oncogene. D, E: CRIP1 expression levels in the GSE24006 and GSE63270 databases, respectively.

Figure 2

CRIP1 expression was up-regulated in AML patients compared with controls (P = 0.045).

Figure 3

Expression of CRIP1 in initial diagnosis (ID), complete remission (CR) and relapsed AML patients receiving induction therapy. CRIP1 expression was increased in AML patients achieved CR than ID patients (P < 0.001). CRIP1 expression was decreased in AML patients achieved CR compared with relapsed AML patients (P = 0.013).

Figure 4

Changes of CRIP1 expression in follow-up AML patients (n = 9) from the initial diagnosis (ID) to complete remission (CR). CRIP1 expression significantly decreased after CR (P = 0.005).

Figure 5

Overall survival (OS) and leukemia-free survival (LFS) between CRIP1 ^high and CRIP1 ^low groups. A, B: Whole-cohort AML patients; CRIP1 ^high group had a shorter OS than those CRIP1 ^low group in the whole-cohort AML patients (P = 0.013). C, D: Non-M3 AML patients; there was a trend that patients with CRIP1 ^high group had a shorter OS than those in CRIP1 ^low group in no-M3 AML (P = 0.057). E, F: CN-AML patients; CRIP1 ^high group had a shorter OS and LFS than those CRIP1 ^low group in CN-AML patients (P < 0.05).

Figure 6

Overall survival and disease-free survival between CRIP1 ^high and CRIP1 ^low group among CN-AML patients in the TCGA cohort (P = 0.010 and 0.008, respectively).

Figure 7

The impact of CRIP1 expression on overall survival in CN-AML by bioinformatics analysis. An independent cohort of CN-AML patients was obtained from Gene Expression Omnibus data (http://www.ncbi.nlm.nih.gov/geo/; accession number GSE12417). Survival analysis was performed through the online web tool Genomicscape (http://genomicscape.com/microarray/survival.php). A: Affy U133 plus 2; B: Affy U133A.

Figure 8

The genomic coordinates (GC) of CRIP1 promoter region CpG island and primer locations. A. The panel plots the GC content as a percentage of the total. Each vertical bar in the bottom panel represents the presence of a CpG dinucleotide. Black horizontal bars indicate regions amplified by MSP primer pairs and BSP primer pairs. This figure was created using CpGplot (http://emboss.bioinformatics.nl/cgibin/emboss/cpgplot) and Methyl Primer Express v1.0 software. TSS: transcription start site; MSP: methylation-specific PCR; BSP: bisulfite sequencing PCR. B. Relative methylation level of CRIP1 promoter in AML patients and controls. C. Methylation density of CRIP1 promoter in AML patients and controls. Methylation density was determined by BSP. White cycle: unmethylated CpG dinucleotide, Black cycle: methylated CpG dinucleotide. C1 and C2: controls; P1 and P2: AML patients.

Figure 9

Correlation between CRIP1 methylation and CRIP1 expression in our group (A; R = -0.027, P = 0.855) and TCGA datasets (B; R = -0.014, P = 0.858).