Genome-wide analysis identifies critical DNA methylations within NTRKs genes in colorectal cancer

Abstract

Background: Neurotrophic tropomyosin receptor kinases (NTRKs) are a gene family function as oncogene or tumor suppressor gene in distinct cancers. We aimed to investigate the methylation and expression profiles and prognostic value of NTRKs gene in colorectal cancer (CRC).

Methods: An analysis of DNA methylation and expression profiles in CRC patients was performed to explore the critical methylations within NTRKs genes. The methylation marker was validated in a retrospectively collected cohort of 229 CRC patients and tested in other tumor types from TCGA. DNA methylation status was determined by quantitative methylation-specific PCR (QMSP).

Results: The profiles in six CRC cohorts showed that NTRKs gene promoter was more frequently methylated in CRC compared to normal mucosa, which was associated with suppressed gene expression. We identified a specific methylated region within NTRK3 promoter targeted by cg27034819 and cg11525479 that best predicted survival outcome in CRC. NTRK3 promoter methylation showed independently predictive value for survival outcome in the validation cohort (P = 0.004, HR 2.688, 95% CI [1.355, 5.333]). Based on this, a nomogram predicting survival outcome was developed with a C-index of 0.705. Furthermore, the addition of NTRK3 promoter methylation improved the performance of currently-used prognostic model (AIC: 516.49 vs 513.91; LR: 39.06 vs 43.64, P = 0.032). Finally, NTRK3 promoter methylation also predicted survival in other tumors, including pancreatic cancer, glioblastoma and stomach adenocarcinoma.

Conclusions: This study highlights the essential value of NTRK3 methylation in prognostic evaluation and the potential to improve current prognostic models in CRC and other tumors.

Keywords: Colorectal cancer; Methylation; NTRK3; NTRKs; Prognosis.

Fig. 1

The flow diagram of cohort analyses. The Fred Hutchinson Cancer Research Center (FHCRC) cohort was an in-house retrospective cohort with 64 colorectal cancer (CRC) samples and 41 normal colon samples. The DNA methylation profile of FHCRC cohort was used to identify the differentially methylated probes (DMPs) within NTRKs genes between tumor and normal samples. We further used the clinical information, DNA methylation and mRNA expression profiles of TCGA-COAD & READ, GSE83889, GSE39582 and GSE87211 cohorts that prospectively enrolled patients with CRC to screen the critical methylated CpG sites within NTRKs genes. The SYSU (Sun Yat-sen University) cohort was an in-house retrospective cohort of 229 patients diagnosed with CRC that was used to validate the identified critical methylated CpGs. The external cohort is a set of 23 tumor types from TCGA, which was used to demonstrate the significance of identified critical methylated CpGs in pan-cancer

Fig. 2

The methylation and expression profiles of NTRKs gene in CRC. a–c. The distribution and correlation of methylation and expression profiles of NTRK1, NTRK2 and NTRK3 gene in CRC and normal mucosa tissue. The heatmaps showed the results of probe-dimensional hierarchical clustering analysis of FHCRC cohort based on β values of all probes within NTRK1 (a, left), NTRK2 (b, left), and NTRK3 (c, left). Each row represented a probe and each column represented a CRC or normal mucosa sample in the heatmap. Probes targeting promoter region were annotated on heatmap left. The red lines annotated in the heatmap for NTRK3 indicated cg27034819 (top) and cg11525479 (bottom). NTRKs gene expression signature in each cohort showed suppressed mRNA expression of NTRK2 (b, middle) and NTRK3 (c, middle) in CRC tissues compared with normal mucosa tissue. Differences between CRC tissue and normal mucosa tissue were assessed with student t-test, and P values summarized with asterisks were shown in the upper space of the plot (***P < 0.001). The Spearman correlation analysis between mean promoter methylation of gene expression in each NTRK gene was shown in right panel. d Volcano plot of statistical significance against hazard ratio for DFS among all CpG sites within NTRKs gene targeted by 450K microarray probes. The top-ranked significantly probe was annotated according to the P values in the univariate Cox analyses. See Additional file 1 for the full results of univariate Cox analysis relevant to this panel. e. A QMSP assay was developed using primers and probe targeting cg27034819–cg11525479 region

Fig. 3

Prognostic significance of NTRK3 promoter methylation. a, b Kaplan–Meier curves of disease-free survival according to NTRK3 promoter methylation in CRC. Kaplan–Meier curves for the whole validation cohort (a) and stage I-III subgroup (b) were shown. The P value for each log-rank test was presented in the plots. Number at risk showed the quantity of CRC patients with NTRK3 hyper- or hypo- methylation and among them the quantity of survivors or dead patients respectively. c, d A nomogram and calibration curve for predicting DFS in CRC. A nomogram to predict individual patient-level 3-year, and 5-year DFS based on clinicopathological risk factors and NTRK3 methylation (c). Calibration plots for the validation sample of the above nomogram (d). Actual DFS statue measured via Kaplan–Meier analysis is shown on the Y-axis, and the nomogram-predicted probability of DFS statue is shown on the X-axis. The average nomogram-predicted probability of DFS was plotted against actually observed DFS estimated by Kaplan–Meier. 95% confidence intervals of the Kaplan–Meier estimates are indicated with vertical lines. Grayline indicates the reference line, showing where an ideal nomogram would lie. Instructions for users: Locate the status on each variable axis, and draw a straight line up to the Points axis to determine how many points toward risk the patient should receive from each variable. Sum the points and locate this number on the Total Points axis. Draw a straight line down from the total points to the 3-year or 5-year DFS Probability axis to ascertain the patient’s specific possibility of maintaining DFS until 3 or 5 years. e, f Pan-cancer analysis of the prognostic significance of NTRK3 promoter methylation. The forest plots showed the values of the HR and CI for the prediction of the survival outcomes in univariate Cox analysis for the methylation of cg27034819 (e) and cg11525479 (f) in 23 TCGA cancer types. The x-axes presenting HRs were log2-scaled