Establishment of permutation for cancer risk estimation in the urothelium based on genome-wide DNA methylation analysis

Abstract

The aim of this study was to establish permutation for cancer risk estimation in the urothelium. Twenty-six samples of normal control urothelium obtained from patients without urothelial carcinomas (C), 47 samples of non-cancerous urothelium without noticeable morphological changes obtained from patients with urothelial carcinomas (N), and 46 samples of the corresponding cancerous tissue (T) in the learning cohort and 64 N samples in the validation cohort, i.e. 183 tissue samples in total, were analyzed. Genome-wide DNA methylation analysis was performed using the Infinium HumanMethylation 450K BeadChip, and DNA methylation levels were verified using pyrosequencing and MassARRAY. Amplicon sequencing was performed using the GeneRead DNAseq Targeted Panels V2. Although N samples rarely showed genetic mutations or copy number alterations, they showed DNA methylation alterations at 2502 CpG sites compared to C samples, and such alterations were inherited by or strengthened in T samples, indicating that DNA methylation alterations may participate in field cancerization in the urothelium. Receiver operating characteristic curve analysis confirmed the feasibility of cancer risk estimation to identify urothelium at the precancerous stage by DNA methylation quantification. Cancer risk estimation permutation was established using a combination of two marker CpG loci on the HOXC4, TENM3 and TLR1 genes (sensitivity and specificity 96–100%). Among them, the diagnostic impact of 10 patterns of permutation was successfully validated in the validation cohort (sensitivity and specificity 94–98%). These data suggest that cancer risk estimation using procedures such as urine tests during health checkups might become applicable for clinical use.

Establishment of diagnostic criteria for upper urinary tract urothelial carcinoma based on genome-wide DNA methylation analysis

“…Even in this study, the DNA methylation profile of N samples tended to differ from that of C samples on the basis of PCA and hierarchical clustering ( Figure 1a, b) . This finding is consistent with our previous data [ 10 , 15 ].…”

“…It is well known that aberrant DNA methylation participates in even the early and precancerous stages in different organs [ 29 , 30 ]. In particular, we have revealed that DNA methylation levels at numerous CpG sites were actually altered in non-cancerous urothelia showing no marked histological changes, but which were clearly at the precancerous stage due to field cancerization, compared to normal urothelia; thus, DNA methylation abnormalities at the precancerous stage can predict future risk or urothelial carcinogenesis [ 15 ]. Even in this study, the DNA methylation profile of N samples tended to differ from that of C samples on the basis of PCA and hierarchical clustering ( Figure 1a, b) .…”

“…DNA methylation alterations are one of the most important epigenomic changes resulting in chromosomal instability and aberrant expression of tumour-related genes [ 6–8 ]. Previous studies by ourselves [ 9–11 ] and other groups [ 12 , 13 ], including the study by The Cancer Genome Atlas (TCGA) [ 14 ], have revealed distinct differences in DNA methylation profiles between non-cancerous urothelia and UCs, suggesting that DNA methylation alterations can become a diagnostic parameter for UC detection [ 15 ]. Moreover, since DNA methylation alterations during carcinogenesis are stably maintained on the DNA double-strand by covalent bonding through a maintenance methylation mechanism involving DNA methyltransferase (DNMT1) [ 16 , 17 ], such alterations can be regarded as a stable indicator for cancer diagnostics, differing from abnormalities of mRNA or protein expression that are easily affected by the microenvironment of cancer cells.…”

Methylation in HOX Clusters and Its Applications in Cancer Therapy

“…Most genes from the HOXC cluster have been identified as hypermethylated in cancer (HOXC4, C5, C6, C8, C9) [48,[115][116][117]. Methylated regions in a gene collection that includes HOXC4 were considered important in estimating cancer risk in urothelium [117] and as part of a prognostic signature predicting survival in patients with oral squamous cell carcinoma [66]. The role of HOXC8 in breast cancer, in which silencing seems to interfere with the self-renewal, differentiation and transformation of breast cancer stem cells, is also instigated by promoter hypermethylation [48].…”

Clinical implications of next‐generation sequencing‐based panel tests for malignant ovarian tumors

“…We identified cancer-specific somatic gene alterations, such as single nucleotide variations (SNVs), insertions/ deletions (indels), and copy number variations (CNVs), as previously described. 7 Furthermore, the tumor mutation burden (TMB) and copy number alterations (CNAs) were calculated using these data. The detailed method used for counting CNA is as follows; for calculating the baseline data used for count correction per amplicon, the number of leads sequenced in each of the 160-panel amplicon probe design domains was counted to calculate the reads per million (RPM) value (ie, the number of leads per one million sequence leads).…”