Relative Telomere Length Change in Colorectal Carcinoma and Its Association with Tumor Characteristics, Gene Expression and Microsatellite Instability.

We compared tumor and adjacent normal tissue samples from 165 colorectal carcinoma (CRC) patients to study change in relative telomere length (RTL) and its association with different histological and molecular features. To measure RTL, we used a Luminex-based assay. We observed shorter RTL in the CRC tissue compared to paired normal tissue (RTL 0.722 ± SD 0.277 vs. 0.809 ± SD 0.242, p = 0.00012). This magnitude of RTL shortening (by ~0.08) in tumor tissue is equivalent to RTL shortening seen in human leukocytes over 10 years of aging measured by the same assay. RTL was shorter in cancer tissue, irrespective of age group, gender, tumor pathology, location and microsatellite instability (MSI) status. RTL shortening was more prominent in low-grade CRC and in the presence of microsatellite instability (MSI). In a subset of patients, we also examined differential gene expression of (a) telomere-related genes, (b) genes in selected cancer-related pathways and (c) genes at the genome-wide level in CRC tissues to determine the association between gene expression and RTL changes. RTL shortening in CRC was associated with (a) upregulation of DNA replication genes, cyclin dependent-kinase genes (anti-tumor suppressor) and (b) downregulation of "caspase executor", reducing apoptosis.

Interaction between Microsatellite Instability (MSI) and Tumor DNA Methylation in the Pathogenesis of Colorectal Carcinoma.

In colorectal cancer (CRC), the role of microsatellite instability (MSI) is well known. In a genome-wide scale, for the first time, we explored whether differential methylation is associated with MSI. We analyzed 250 paired samples from 125 CRC patients (m = 72, f = 53) at different stages. Of them, 101 had left-sided CRC, 30 had MSI, 34 had somatic mutation in KRAS proto-oncogene (KRAS), and 6 had B-Raf proto-oncogene (BRAF) exon 15p.V600E mutation. MSI was more frequent in right-sided tumors (54% vs. 17%, p = 0.003). Among the microsatellite stable (MSS) CRC, a paired comparison revealed 1641 differentially methylated loci (DML) covering 686 genes at FDR 0.001 with delta beta ≥ 20%. Similar analysis in MSI revealed 6209 DML covering 2316 genes. ANOVA model including interaction (Tumor*MSI) revealed 23,322 loci, where the delta beta was different among MSI and MSS patients. Our study shows an association between MSI and tumor DNA methylation in the pathogenesis of CRC. Given the interaction seen in this study, it may be worth considering the MSI status while looking for methylation markers in CRC. The study also indicates an opportunity for potential use of certain immune checkpoint inhibitors (CTLA4 and HAVCR2 inhibitors) in CRC with MSI.

A genome-wide study of cytogenetic changes in colorectal cancer using SNP microarrays: opportunities for future personalized treatment.

In colorectal cancer (CRC), chromosomal instability (CIN) is typically studied using comparative-genomic hybridization (CGH) arrays. We studied paired (tumor and surrounding healthy) fresh frozen tissue from 86 CRC patients using Illumina's Infinium-based SNP array. This method allowed us to study CIN in CRC, with simultaneous analysis of copy number (CN) and B-allele frequency (BAF)--a representation of allelic composition. These data helped us to detect mono-allelic and bi-allelic amplifications/deletion, copy neutral loss of heterozygosity, and levels of mosaicism for mixed cell populations, some of which can not be assessed with other methods that do not measure BAF. We identified associations between CN abnormalities and different CRC phenotypes (histological diagnosis, location, tumor grade, stage, MSI and presence of lymph node metastasis). We showed commonalities between regions of CN change observed in CRC and the regions reported in previous studies of other solid cancers (e.g. amplifications of 20q, 13q, 8q, 5p and deletions of 18q, 17p and 8p). From Therapeutic Target Database, we identified relevant drugs, targeted to the genes located in these regions with CN changes, approved or in trials for other cancers and common diseases. These drugs may be considered for future therapeutic trials in CRC, based on personalized cytogenetic diagnosis. We also found many regions, harboring genes, which are not currently targeted by any relevant drugs that may be considered for future drug discovery studies. Our study shows the application of high density SNP arrays for cytogenetic study in CRC and its potential utility for personalized treatment.

Interpretation of genome-wide infinium methylation data from ligated DNA in formalin-fixed, paraffin-embedded paired tumor and normal tissue.

BACKGROUND: Formalin-fixed, paraffin-embedded (FFPE) samples are a highly desirable resource for epigenetic studies, but there is no suitable platform to assay genome-wide methylation in these widely available resources. Recently, Thirlwell et al. (2010) have reported a modified ligation-based DNA repair protocol to prepare FFPE DNA for the Infinium methylation assay. In this study, we have tested the accuracy of methylation data obtained with this modification by comparing paired fresh-frozen (FF) and FFPE colon tissue (normal and tumor) from colorectal cancer patients. We report locus-specific correlation and concordance of tumor-specific differentially methylated loci (DML), both of which were not previously assessed. METHODS: We used Illumina's Infinium Methylation 27K chip for 12 pairs of FF and 12 pairs of FFPE tissue from tumor and surrounding healthy tissue from the resected colon of the same individual, after repairing the FFPE DNA using Thirlwell's modified protocol. RESULTS: For both tumor and normal tissue, overall correlation of ß values between all loci in paired FF and FFPE was comparable to previous studies. Tissue storage type (FF or FFPE) was found to be the most significant source of variation rather than tissue type (normal or tumor). We found a large number of DML between FF and FFPE DNA. Using ANOVA, we also identified DML in tumor compared to normal tissue in both FF and FFPE samples, and out of the top 50 loci in both groups only 7 were common, indicating poor concordance. Likewise, while looking at the correlation of individual loci between FFPE and FF across the patients, less than 10% of loci showed strong correlation (r ≥ 0.6). Finally, we checked the effect of the ligation-based modification on the Infinium chemistry for SNP genotyping on an independent set of samples, which also showed poor performance. CONCLUSION: Ligation of FFPE DNA prior to the Infinium genome-wide methylation assay may detect a reasonable number of loci, but the numbers of detected loci are much fewer than in FF samples. More importantly, the concordance of DML detected between FF and FFPE DNA is suboptimal, and DML from FFPE tissues should be interpreted with great caution.