Faecal ribosomal protein L19 is a genetic prognostic factor for survival in colorectal cancer

Abstract

Ribosomal proteins are encoded by a gene family, members of which are overexpressed in human cancers. Many of them have been found, using oligonucleotide microarray hybridization, to be differentially expressed in the faeces of patients with various stages of colorectal cancer (CRC). The gene encoding ribosomal protein L19 (RPL19), a prognostic marker for human prostate cancer, is differentially expressed in CRC patients. Measurement of faecal RPL19 mRNA might improve prognostic prediction for CRC patients. Using quantitative real-time reverse transcription PCR, levels of RPL19 mRNA were detected in samples of colonic tissues from 44 CRC patients, in the faeces of 54 CRC patients and 15 controls, and in 11 colonic cell lines. Seven of 24 patients with late-stage CRC (Dukes' stages C and D) expressed over 2-fold more RPL19 in colonic tumour tissues than in corresponding normal tissues (P= 0.038). The mean faecal RPL19 mRNA levels of late-staged patients were higher than those of controls (P= 0.003) and early-staged patients (P= 0.008). Patients with both high serum levels of carcinoembryonic antigen (CEA; > 5 ng/mL) and high-faecal RPL19 mRNA (> or =0.0069) had higher risk (odds ratio, 8.0; P= 0.015) and lower overall 48-month survival (33.8 +/- 13.7%, P= 0.013). Oligonucleotide microarray hybridization analysis of faecal molecules identified gene transcripts differentially present in faeces. In conclusion, faecal RPL19 expression is associated with advanced tumour stages and addictive to serum CEA in predicting prognosis of CRC patients.

Fig 1

Differentially expressed RPs in the faeces of 11 CRC patients and five normal controls. Forty-six genes for RPs and their related transcripts were generated with statistically significant difference (fold change ≥2, P < 0.01) from pooling each comparison and grouped by average-linkage hierarchical clustering. Each row represents a gene and each column a sample. (A) A dendrogram depicting the groups of the cases. A group cluster depicts the cases with a similar pattern of gene expressions. Group I: five patients, one Dukes' stage B (patient 08), one stage C (patient 06) and three stage D (patients 11, 05 and 09). Group II: two normal control pools, NP01 and NP02; and six patients, two Dukes' stage A (patients 07 and 04), two stage B (patients 10 and 01), two stage C (patients 02 and 03). NP01, pooled by two healthy men; NP02, pooled by three healthy women. (B) The classification of 46 genes. A region cluster depicts the genes based on the similarity between their expressions in cases. High expression is shown in red, low expression in green. Region A (orange) contained 22 genes whose expressions were up in group I (light blue) and down in group II (pink). Region B (violet) contained 24 genes whose expressions were down in group I and up in group II. *RPL19 (X63527), the transcript for ribosomal protein L19.

Fig 2

Relative cellular RPL19 expression levels from colon cell lines with quantitative real-time PCR. Quantitative real-time PCR was performed using LightCycler 2.0 from 11 colonic cell lines. The expression level of RPL19 mRNA was normalized by dividing it by that for 18s rRNA for each line. Bar 1, CCD-18Co; bar 2, CCD-112CoN; bar 3, SW 1116; bar 4, SW480; bar 5, HCT 116; bar 6, DLD-1; bar 7, LoVo; bar 8, Caco-2; bar 9, HT-29; bar 10, COLO 205; bar 11, T84.

Fig 3

Relative faecal RPL19 expression levels from different populations with quantitative real-time PCR. Quantitative real-time PCR was performed using LightCycler 2.0 from healthy individuals (n= 15), patients with early-stage colorectal cancer (CRC) (Dukes' stages A and B; n= 26), and patients with late-stage CRC (Dukes' stages C and D; n= 28). The expression level of RPL19 mRNA was normalized by dividing it by that for 18s rRNA for each faecal sample. A significant difference (P < 0.05) determined by Mann-Whitney U test is directly indicated as P value. The horizontal lines represent the median values.

Fig 4

Kaplan–Meier survival curves of CRC patients based on the levels of serum CEA and relative faecal RPL19 expression. Patients with low-serum CEA and low-faecal RPL19 expression (CEA⁻/RPL19⁻): n= 18; overall 48-month survival: 70.6 ± 17.0%. Patients with low serum CEA and high-faecal RPL19 expression (CEA⁻/RPL19⁺): n= 14; overall 48-month survival: 44.4 ± 21.1%. Patients with high-serum CEA and high-faecal RPL19 expression (CEA⁺/RPL19⁺): n= 13; overall 48-month survival: 33.8 ± 13.7%. RPL19⁻, relative faecal RPL19 expression value <0.0069 (mean value of 26 patients with early-stage CRC); RPL19⁺, relative faecal RPL19 expression value ≥ 0.0069. The P-value was determined using the log rank test.