Clinical and epigenetic features of colorectal cancer patients with somatic POLE proofreading mutations

Abstract

Background: Mutations in the POLE gene result in an ultra-hypermutated phenotype in colorectal cancer (CRC); however, the molecular characterisation of epigenetic alterations remains unclear. We examined the genetic and epigenetic profiles of POLE-mutant CRC to elucidate the clinicopathological features of the associated genetic and epigenetic alterations.

Results: Tumour tissues (1,013) obtained from a cohort of patients with CRC were analysed to determine associations between the proofreading domain mutations of POLE with various clinicopathological variables, microsatellite instability (MSI) status, BRAF and KRAS mutations, and the methylation status of key regions of MLH1, MGMT, and SFRP2 promoters by calculating the methylation scores (range 0-6). Only four cases (0.4%) exhibited pathogenic POLE hotspot mutations (two p.P286R [c.857C > G], one p.V411L [c.1231G > C], and p.S459F [c.1376C > T] each), which were mutually exclusive to BRAF and KRAS mutations and MSI. CRC patients were divided into four subgroups: patients with POLE mutations (POLE, 0.4%, n = 4), patients with both MSI and extensive methylation in MLH1 (MSI-M, 2.9%, n = 29), patients with MSI but no extensive methylation in MLH1 (MSI-U, 3.6%, n = 36), and patients without MSI (non-MSI, 93.2%, n = 944). The POLE group was younger at diagnosis (median 52 years, P < 0.0001), with frequent right-sided tumour localisation (frequency of tumours located in the right colon was 100%, 93.1%, 36.1%, and 29.9% in POLE, MSI-M, MSI-U, and non-MSI, respectively; P < 0.0001), and was diagnosed at an earlier stage (frequency of stages I-II was 100%, 72.4%, 77.8%, and 46.6% in POLE, MSI-M, MSI-U, and non-MSI, respectively, P < 0.0001). The mean methylation score in POLE was not different from that in MSI-U and non-MSI, but the methylation signature was distinct from that of the other subgroups. Additionally, although the examined number of POLE-mutant tumours was small, the number of CD8-positive cells increased in tumours with partial methylation in the MLH1 gene.

Conclusions: CRC patients with POLE proofreading mutations are rare. Such mutations are observed in younger individuals, and tumours are primarily located in the right colon. Diagnosis occurs at an earlier stage, and distinct epigenetic alterations may be associated with CD8 cell infiltration.

Keywords: Colorectal cancer; MLH1; Methylation; Microsatellite instability; POLE.

Fig. 1

The STROBE diagram of the colorectal cancer patient cohort. *Patients with synchronous multiple cancers based on the most advanced tumour lesion were subsequently confirmed pathologically for further molecular studies

Fig. 2

Detection of POLE proofreading mutations. Among 1,070 CRC tissues analysed, pathogenic somatic POLE proofreading mutations were detected in four CRCs (two cases were p.P286R [c.857C > G], one was p.V411L [c.1231G > C], and one was p.S459F [c.1376C > T]) and DNA from their corresponding normal mucosa displayed no germline mutations

Fig. 3

Methylation analysis of the promoter region in the MLH1 gene. a Schematic depiction of two regions (AB and C region) of the MLH1 promoter for methylation, and results of a panel of representative fluorescent bisulphite PCR following restriction enzyme analysis. AB and C region in this study link to A plus B and C plus D region, defined by Deng and colleagues [18], respectivily.  Methylated samples had the new fragment cleaved by the restriction enzyme (black triangles). White triangles represent unmethylated alleles. M and U denote methylated and unmethylated, respectively. A grey bar denotes an untranslated exon. b The frequencies of MLH1 promoter methylation status among the POLE, MSI, and non-MSI groups. Extensive methylation is defined as 5.0% or more methylation on both AB and C regions. Partial methylation is defined as 5.0% or more methylation on AB region only. c The frequencies of MLH1 promoter methylation status according to MMR protein expression status. dMLH1, dMSH2, and dMSH6 denote deficiency of MLH1, MSH2, and MSH6, respectively. pMMR denotes proficiency of all MMR proteins. d The final classification of 1,013 CRCs according to POLE mutation, MSI, and MLH1 promoter methylation status. MSI-M denotes MSI tumours with extensive MLH1 methylation. MSI-U denotes MSI tumours without extensive MLH1 methylation. The number in each column denotes the number of samples classified in each category divided by MLH1 methylation status

Fig. 4

Methylation analysis of the promoter region in the MGMT and SFRP2 gene. a Schematic depiction of two regions (Mp and Eh region) of the MGMT promoter for methylation, and results of a panel of representative fluorescent bisulphite PCR following restriction enzyme analysis. Methylated samples had the new fragment cleaved by the restriction enzyme (black triangles). White triangles represent unmethylated alleles. M and U denote methylated and unmethylated, respectively. A grey bar denotes an untranslated exon. b The frequencies of MGMT promoter methylation status among the POLE, MSI-M, MSI-U, and non-MSI groups. Extensive methylation is defined as 5.0% or more methylation on both Mp and Eh regions. Partial methylation is defined as 5.0% or more methylation on either Eh or Mp region. c Schematic depiction of two regions (R1 and R2 region) of the SFRP2 promoter for methylation, and results of a panel of representative fluorescent bisulphite PCR following restriction enzyme analysis. Methylated samples had the new fragment cleaved by the restriction enzyme (black triangles). White triangles represent unmethylated alleles. M and U denote methylated and unmethylated, respectively. A black bar denotes translated exon. d The frequencies of SFRP2 promoter methylation status among the POLE, MSI-M, MSI-U, and non-MSI groups. Extensive methylation is defined as 5.0% or more methylation on both R1 and R2 region. Partial methylation is defined as 5.0% or more methylation on either R1 or R2 region. The number in each column denotes the number of samples classified in each category divided by MGMT or SFRP2 methylation status

Fig. 5

Features of POLE-mutant CRCs. a Mean methylation score in various subgroups of CRCs categorised by POLE mutation, MSI, and MLH1 promoter methylation status. The mean methylation score in each subset was calculated based on the MLH1, MGMT, and SFRP2 gene (six loci). In the red box plot diagrams, the horizontal line within each box represents the median; the limits of each box are the interquartile ranges, the whiskers are the maximum and minimum values, and the green horizontal bar within each box depicts the mean value. The numbers under the green horizontal bar denote the mean methylation score. The P values above the square panels are statistical differences among any two individual groups calculated by the Steel–Dwass test. b Representative immunohistochemical images for the cytotoxic T-cell marker CD8 (brown) in POLE-mutant CRC samples. c Association among age at diagnosis, MLH1-AB region methylation status, and the number of CD8-positive ( +) cells per HPF in POLE-mutant CRCs. The P value is calculated by the Kruskal–Wallis test. Red circles denote patients with POLE proofreading mutations. HPF; high-power field (× 400)