Methylation profiling of rectal cancer identifies novel markers of early-stage disease.

BACKGROUND: Radical surgery is the de facto treatment for early rectal cancer. Conservative surgery with transanal endoscopic microsurgery can achieve high rates of cure but the histopathological measures of outcome used to select local treatment lack precision. Biomarkers associated with disease progression, particularly mesorectal nodal metastasis, are urgently required. The aim was to compare patterns of gene-specific hypermethylation in radically excised rectal cancers with histopathological stage. METHODS: Locus-specific hypermethylation of 24 tumour suppressor genes was measured in 105 rectal specimens (51 radically excised adenocarcinomas, 35 tissues adjacent to tumour and 19 normal controls) using the methylation-specific multiplex ligation-dependent probe assay (MS-MLPA). Methylation values were correlated with histopathological indices of disease progression and validated using bisulphite pyrosequencing. RESULTS: Five sites (ESR1, CDH13, CHFR, APC and RARB) were significantly hypermethylated in cancer compared with adjacent tissue and normal controls (P < 0·050). Methylation at these sites was higher in Dukes' A than Dukes' 'D' cancers (P = 0·013). Methylation at two sites (GSTP1 and RARB) was individually associated with localized disease (N0 and M0 respectively; P = 0·006 and P = 0·008). Hypermethylation of at least two of APC, RARB, TIMP3, CASP8 and GSTP1 was associated with early (N0 M0) disease (N0, P = 0·002; M0, P = 0·044). Methylation levels detected by MS-MLPA and pyrosequencing were concordant. CONCLUSION: Locus-specific hypermethylation was more prevalent in early- than late-stage disease. Hypermethylation of two or more of a panel of five tumour suppressor genes was associated with localized disease.

Wnt signalling in adenomas of familial adenomatous polyposis patients.

BACKGROUND: Epigenetic silencing of Wnt antagonists and expression changes in genes associated with Wnt response pathways occur in early sporadic colorectal tumourigenesis, indicating that tumour cells are more sensitive to Wnt growth factors and respond differently. In this study, we have investigated whether similar changes occur in key markers of the Wnt response pathways in the genetic form of the disease, familial adenomatous polyposis (FAP). METHODS: We investigated epigenetic and expression changes using pyrosequencing and real-time RT-PCR in samples from seven patients without neoplasia, and matched normal and tumour tissues from 22 sporadic adenoma and 14 FAP patients. RESULTS: We found that 17 out of 24 (71%) FAP adenomas were hypermethylated at sFRP1, compared with 20 out of 22 (91%) of sporadic cases. This was reflected at the level of sFRP1 transcription, where 73% of FAP and 100% of sporadic cases were down-regulated. Increased expression levels of c-myc and FZD3 were less common in FAP (35 and 46% respectively) than sporadic tumours (78 and 67% respectively). CONCLUSION: Overall, the changes in expression and methylation were comparable, although the degree of change was generally lower in the FAP adenomas. Molecular heterogeneity between multiple adenomas from individual FAP patients may reflect different developmental fates for these premalignant tumours.

Reorganisation of Wnt-response pathways in colorectal tumorigenesis.

In most colorectal tumours, APC mutation stabilises beta-catenin and mimics elements of Wnt growth factor signalling, but the high frequency of epigenetic loss of Wnt antagonists indicates an additional role for ligand-mediated Wnt signalling. Here, we have investigated the expression of key components of beta-catenin-independent Wnt response pathways to determine whether their profiles change during the transition from normal mucosa to colorectal adenomas. Transcription of the Wnt/planar cell polarity pathway determinant NKD1 (naked cuticle homologue 1) was induced in adenomas by a median 135-fold and in cancers by 7.4-fold. While some Frizzleds (FZDs) were downregulated in adenomas, the Wnt/Ca(2+) receptors FZD3 and FZD6 were induced by a median factor of 6.5 and 4.6, respectively. Naked cuticle homologue 1, FZD3 and FZD6 expression were coordinated in pre-malignant disease, but this relationship was lost in invasive cancers, where FZD induction was seen less frequently. Naked cuticle homologue 1 expression was associated with nuclear localisation of phospho-c-Jun in adenomas. In cultured cells, NKD1 transcription was induced by lithium chloride but FZD3 expression required Wnt growth factor treatment. These data show that Wnt responses are consistently directed towards both beta-catenin-independent routes in early colorectal tumorigenesis and elements of this are retained in more advanced cancers. These beta-catenin-independent Wnt signalling pathways may provide novel targets for chemoprevention of early colorectal tumours.

The Wnt antagonist sFRP1 is downregulated in premalignant large bowel adenomas.

Our previous studies have implicated the Wnt antagonist, sFRP1, as a tumour suppressor gene in advanced colorectal cancer. In this study, we set out to investigate the relationship between sFRP1 expression and large bowel adenomas, a precursor of colorectal cancer. The induction of beta-catenin/TCF mediated transcription is both a frequent early event in colorectal neoplasia, and a key downstream effect of wnt growth factor signalling. Lithium treatment of a small bowel mucosal cell line (FHs 74 int) induced sFRP1 within 8 h, indicating that this gene is positively regulated by beta-catenin, contrasting with the suppression of sFRP1 expression, we saw previously in advanced colorectal cancers. We therefore investigated a series of 12 adenomas and matched large bowel mucosa samples. Real-time RT-PCR analysis showed a reduction in sFRP1 expression in all 12 dysplastic lesions (median 485-fold, IQR 120- to 1500-fold), indicating factors other than beta-catenin influence sFRP1 levels. In a second series of 11 adenomas, we identified methylation of the sFRP1 promotor region in all 11 samples, and this was increased compared with the surrounding normal mucosa in seven cases. Immunohistochemical analysis using a polyclonal antibody supported these findings, with sFRP1 expression reduced in many of the adenoma samples examined. sFRP1 staining in normal mucosa adjacent to the dysplastic tissue was also reduced compared with the normal controls, suggesting that sFRP1 expression may be suppressed in a field of mucosa rather than in individual cells. This study identifies sFRP1 inactivation at the premalignant stage of colorectal cancer development, indicating that these pathways may be useful targets for chemoprevention strategies in this common solid tumour.

Mapping of genes and transcribed sequences in a gene rich 400-kb region on human chromosome 11p15.1-->p14.

We have identified a number of transcribed sequences within a 400-kb interval on chromosome 11p15.1--> p14. Six genes and 13 novel transcripts including ESTs, cDNAs and exons have been identified and assigned to this region. Comparison of mRNA sequence with genomic sequence has enabled us to determine the exon/intron structure of four of the genes (NUCB2, PIK3C2A, RPS13 and OR7E14P).

A maternally methylated CpG island in KvLQT1 is associated with an antisense paternal transcript and loss of imprinting in Beckwith-Wiedemann syndrome.

Loss of imprinting at IGF2, generally through an H19-independent mechanism, is associated with a large percentage of patients with the overgrowth and cancer predisposition condition Beckwith-Wiedemann syndrome (BWS). Imprinting control elements are proposed to exist within the KvLQT1 locus, because multiple BWS-associated chromosome rearrangements disrupt this gene. We have identified an evolutionarily conserved, maternally methylated CpG island (KvDMR1) in an intron of the KvLQT1 gene. Among 12 cases of BWS with normal H19 methylation, 5 showed demethylation of KvDMR1 in fibroblast or lymphocyte DNA; whereas, in 4 cases of BWS with H19 hypermethylation, methylation at KvDMRl was normal. Thus, inactivation of H19 and hypomethylation at KvDMR1 (or an associated phenomenon) represent distinct epigenetic anomalies associated with biallelic expression of IGF2. Reverse transcription-PCR analysis of the human and syntenic mouse loci identified the presence of a KvDMR1-associated RNA transcribed exclusively from the paternal allele and in the opposite orientation with respect to the maternally expressed KvLQT1 gene. We propose that KvDMR1 and/or its associated antisense RNA (KvLQT1-AS) represents an additional imprinting control element or center in the human 11p15.5 and mouse distal 7 imprinted domains.

Bestrophin gene mutations in patients with Best vitelliform macular dystrophy.

Best vitelliform macular dystrophy (VMD2) is an autosomal dominant dystrophy with a juvenile age of onset. Mutations in the Bestrophin gene were shown in patients affected with VMD2. In a mutation study, we made three new and interesting observations. First, we identified possible mutation hotspots within the gene, suggesting that particular regions of the protein have greater functional significance than others. Second, we described a 2-bp deletion in a part of the gene where mutations have not previously been reported; this mutation causes a frameshift and subsequent premature termination of the protein. Finally, we have evidence that some mutations are associated with variable expression of the disease, suggesting the involvement of other factors or genes in the disease phenotype.