Association of Microsatellite Instability and Gene Expression Profile in Colorectal Carcinoma and Potential Implications for Therapy.

Background and Objective: In sporadic colorectal carcinomas (CRC), microsatellite instability (MSI) pathways play important roles. Previously, we showed differences in DNA methylation patterns in microsatellite stable (MSS) colorectal carcinomas and MSI-CRC. In the current study, we explore the similarities and differences in gene expression profiles in MSS and MSI at the gene level and at the pathway level to better understand CRC pathogenesis and/or the potential for therapeutic opportunities. Material and Methods: Seventy-one CRC patients (MSI = 18, MSS = 53) were studied. Paired tumor and adjacent normal tissues were used for genome-wide gene expression assays. Result: At the gene level, we compared the list of differentially expressed genes (fold change (FC) ≥ 3 and FDR < 0.05) in tumor tissues compared to corresponding normal tissue in CRC patients with MSI tumors (190 genes) and MSS tumors (129 genes). Of these, 107 genes overlapped. The list of genes that were differentially expressed in MSI tumors only showed enrichment predominantly in two broad categories of pathways-(a) Inflammation-related pathways including the interleukin-17 (IL-17) signaling pathway, tumor necrosis factor (TNF) signaling pathway, chemokine signaling, nuclear factor kappa B (NFκB) signaling, and cytokine-cytokine interactions, and (b) metabolism-related pathways, including retinol metabolism, steroid hormone biosynthesis, drug metabolism, pentose and glucoronate interconversions, and ascorbate and aldarate metabolism. The genes in inflammation-related pathways were up-regulated whereas genes in metabolism-related pathways were down-regulated in MSI tumor tissue. Pathway-level analysis also revealed similar results confirming the gene enrichment findings. For example, the 150 genes involved in the IL-17 signaling pathway were on average up-regulated by 1.19 fold (CI 1.16-1.21) in MSI compared to 1.14 fold (CI 1.13-1.16) in MSS patients (interaction p = 0.0009). Conclusions: We document an association between MSI status and differential gene expression that broadens our understanding of CRC pathogenesis. Furthermore, targeting one or more of these dysregulated pathways could provide the basis for improved therapies for MSI and MSS CRC.

Pathways Related to Colon Inflammation Are Associated with Colorectal Carcinoma: A Transcriptome- and Methylome-Wide Study.

The association of chronic inflammation with colorectal carcinoma (CRC) development is well known in ulcerative colitis (UC). However, the role of inflammatory changes in sporadic CRC pathogenesis is less widely appreciated. In this study, in the first step using RNA-seq, we identified gene-pathway-level changes in UC-associated CRC (UC CRC, n = 10) and used the changes as a proxy for inflammation in human colon to ask if there were associations of inflammatory pathway dysregulations in sporadic CRC pathogenesis (n = 8). We found down-regulations of several inflammation-related metabolic pathways (nitrogen metabolism, sulfur metabolism) and other pathways (bile secretion, fatty acid degradation) in sporadic CRC. Non-inflammation-related changes included up-regulation of the proteasome pathway. In the next step, from a larger number of paired samples from sporadic CRC patients (n = 71) from a geographically and ethnically different population and using a different platform (microarray), we asked if the inflammation-CRC association could be replicated. The associations were significant even after stratification by sex, tumor stage, grade, MSI status, and KRAS mutation status. Our findings have important implications to widen our understanding of inflammatory pathogenesis of sporadic CRC. Furthermore, targeting of several of these dysregulated pathways could provide the basis for improved therapies for CRC.

A Transcriptome and Methylome Study Comparing Tissues of Early and Late Onset Colorectal Carcinoma.

There is an increase in the incidence of early onset colorectal carcinoma (EOCRC). To better understand if there is any difference in molecular pathogenesis of EOCRC and late onset colorectal carcinoma (LOCRC), we compared the clinical, histological, transcriptome, and methylome profile of paired CRC and healthy colonic tissue from 67 EOCRC and 98 LOCRC patients. The frequency of stage 3 CRC, lymph node involvement, lymphovascular invasion, and perineural invasion was higher in the EOCRC group. Many of the cancer related pathways were differentially expressed in CRC tissue in both EOCRC and LOCRC patients. However, the magnitude of differential expression for some groups of genes, such as DNA damage repair genes and replication stress genes, were significantly less pronounced in the EOCRC group, suggesting less efficient DNA damage repair to be associated with EOCRC. A more marked methylation of "growth factor receptor" genes in LOCRC correlated with a more pronounced down-regulation of those genes in that group. From a therapeutic point of view, more over-expression of fatty acid synthase (FASN) among the LOCRC patients may suggest a better response of FASN targeted therapy in that group. The age of onset of CRC did not appear to modify the response of cis-platin or certain immune checkpoint inhibitors. We found some differences in the molecular pathogenesis in EOCRC and LOCRC that may have some biological and therapeutic significance.

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.

A genome-wide DNA methylation study in colorectal carcinoma.

BACKGROUND: We performed a genome-wide scan of 27,578 CpG loci covering 14,475 genes to identify differentially methylated loci (DML) in colorectal carcinoma (CRC). METHODS: We used Illumina's Infinium methylation assay in paired DNA samples extracted from 24 fresh frozen CRC tissues and their corresponding normal colon tissues from 24 consecutive diagnosed patients at a tertiary medical center. RESULTS: We found a total of 627 DML in CRC covering 513 genes, of which 535 are novel DML covering 465 genes. We also validated the Illumina Infinium methylation data for top-ranking genes by non-bisulfite conversion q-PCR-based methyl profiler assay in a subset of the same samples. We also carried out integration of genome-wide copy number and expression microarray along with methylation profiling to see the functional effect of methylation. Gene Set Enrichment Analysis (GSEA) showed that among the major "gene sets" that are hypermethylated in CRC are the sets: "inhibition of adenylate cyclase activity by G-protein signaling", "Rac guanyl-nucleotide exchange factor activity", "regulation of retinoic acid receptor signaling pathway" and "estrogen receptor activity". Two-level nested cross validation showed that DML-based predictive models may offer reasonable sensitivity (around 89%), specificity (around 95%), positive predictive value (around 95%) and negative predictive value (around 89%), suggesting that these markers may have potential clinical application. CONCLUSION: Our genome-wide methylation study in CRC clearly supports most of the previous findings; additionally we found a large number of novel DML in CRC tissue. If confirmed in future studies, these findings may lead to identification of genomic markers for potential clinical application.