DNA methylation is an epigenetic modification that plays a crucial role in the control of gene expression and chromosome structure in plants and mammalian cells. Multiple types of DNA fingerprinting techniques have been developed and applied to investigate DNA methylation profiles in different experimental settings. One of these techniques, the amplification of intermethylated sites (AIMS) is a simple approach appropriate for genome-wide estimates of DNA methylation and the discovery of specific methylated sequences. AIMS is based on the differential enzymatic digestion of genomic DNA with methylation-sensitive and methylation-insensitive isoschizomers followed by restrained PCR amplification of methylated sequences. This method is appropriate to compare large series of samples and the simultaneous identification of hypo- and hypermethylation events. Applications of AIMS include the study of DNA methylation changes in cancer and aging, and the discovery of DNA methylation in a social insect.

Methylation of the cytosine is the most frequent epigenetic modification of DNA in mammalian cells. In humans, most of the methylated cytosines are found in CpG-rich sequences within tandem and interspersed repeats that make up to 45% of the human genome, being Alu repeats the most common family. Demethylation of Alu elements occurs in aging and cancer processes and has been associated with gene reactivation and genomic instability. By targeting the unmethylated SmaI site within the Alu sequence as a surrogate marker, we have quantified and identified unmethylated Alu elements on the genomic scale. Normal colon epithelial cells contain in average 25 486 +/- 10 157 unmethylated Alu's per haploid genome, while in tumor cells this figure is 41 995 +/- 17 187 (P = 0.004). There is an inverse relationship in Alu families with respect to their age and methylation status: the youngest elements exhibit the highest prevalence of the SmaI site (AluY: 42%; AluS: 18%, AluJ: 5%) but the lower rates of unmethylation (AluY: 1.65%; AluS: 3.1%, AluJ: 12%). Data are consistent with a stronger silencing pressure on the youngest repetitive elements, which are closer to genes. Further insights into the functional implications of atypical unmethylation states in Alu elements will surely contribute to decipher genomic organization and gene regulation in complex organisms.

BACKGROUND: Papillary thyroid cancer (PTC) is the most common type of thyroid cancer. Unlike most cancers, its incidence has dramatically increased in the last decades mainly due to increased diagnosis of indolent PTCs. Adequate risk stratification is crucial to avoid the over-treatment of low-risk patients, as well as the under-treatment of high-risk patients, but the currently available markers are still insufficient. Kallikreins (KLKs) are emergent biomarkers in cancer, but their involvement in PTC is unknown. METHODS: This study analyzed DNA methylation (HumanMethylation arrays) and gene expression (RNA-Seq) of KLKs, BRAF and RAS mutations, and clinical data from four published thyroid cancer data sets including normal and tumor tissues (n = 73, n = 475, n = 20, and n = 82) as discovery, training, and validation series. The C4.5 classification algorithm was used to generate a decision tree. Disease-free survival was estimated using Kaplan-Meier and Cox approaches. Specific analyses were performed using real-time polymerase chain reaction and immunohistochemistry. RESULTS: or RAS mutations. Thus, a decision-tree algorithm was developed based on three KLKs with >80% sensitivity and >95% specificity, identifying BRAF- and RAS-mutated tumors. Notably, tumors lacking these mutations were classified as BRAF- or RAS-like. Most importantly, the KLK algorithm uncovered a novel PTC subtype showing favorable prognostic features. CONCLUSIONS: The KLK algorithm could lead to a new clinically applicable strategy with important implications for the risk stratification of PTC and the management of patients.

Adipose tissue (AT) has a central role in obesity-related metabolic imbalance through the dysregulated production of cytokines and adipokines. In addition to its known risk for cardiovascular disease and diabetes, obesity is also a major risk for cancer. We investigated the impact of obesity for the expression of survivin, an antiapoptotic protein upregulated by adipokines and a diagnostic biomarker of tumor onset and recurrence. In a cross-sectional study of 111 subjects classified by body mass index, circulating levels of survivin and gene expression in subcutaneous AT were significantly higher in obese patients and positively correlated with leptin. Within AT, survivin was primarily detected in human adipocyte-derived stem cells (hASCs), the adipocyte precursors that determine AT expansion. Remarkably, survivin expression was significantly higher in hASCs isolated from obese patients that from lean controls and was increased by proinflammatory M1 macrophage soluble factors including IL-1β. Analysis of survivin expression in hASCs revealed a complex regulation including epigenetic modifications and protein stability. Surprisingly, obese hASCs showed survivin promoter hypermethylation that correlated with a significant decrease in its mRNA levels. Nonetheless, a lower level of mir-203, which inhibits survivin protein translation, and higher protein stability, was found in obese hASCs compared with their lean counterparts. We discovered that survivin levels determine the susceptibility of hASCs to apoptotic stimuli (including leptin and hypoxia). Accordingly, hASCs from an obese setting were protected from apoptosis. Collectively, these data shed new light on the molecular mechanisms governing AT expansion in obesity through promotion of hASCs that are resistant to apoptosis, and point to survivin as a potential new molecular player in the communication between AT and tumor cells. Thus, inhibition of apoptosis targeting survivin might represent an effective strategy for both obesity and cancer therapy.

Silencing of multiple cancer-related genes is associated with de novo methylation of linked CpG islands. Additionally, bivalent histone modification profiles characterized by the juxtaposition of active and inactive histone marks have been observed in genes that become hypermethylated in cancer. It is unknown how these ambiguous epigenetic states are maintained and how they interrelate with adjacent genomic regions with different epigenetic landscapes. Here, we present the analysis of a set of neighboring genes, including many frequently silenced in colon cancer cells, in a chromosomal region at 5q35.2 spanning 1.25 Mb. Promoter DNA methylation occurs only at genes maintained at a low transcriptional state and is characterized by the presence of bivalent histone marks, namely trimethylation of lysines 4 and 27 in histone 3. Chemically induced hyperacetylation and DNA demethylation lead to up-regulation of silenced genes in this locus yet do not resolve bivalent domains into a domain-wide active chromatin conformation. In contrast, active genes in the region become down-regulated after drug treatment, accompanied by a partial loss of chromatin domain boundaries and spreading of the inactive histone mark trimethylated lysine 27 in histone 3. Our results demonstrate that bivalent domains mark the promoters of genes that will become DNA methylated in adult tumor cells to enforce transcriptional silence. These bivalent domains not only remain upon drug induced gene reactivation, but also spread over adjacent CpG islands. These results may have important implications in understanding and managing epigenetic therapies of cancer.

Ziemannâ s supplementary file. Tab-separated, plain text version of the Ziemann et al. [2] supplementary file. (TSV 148 kb)

The aim of this study is to establish an estimation of the global genomic alteration in neuroblastic tumors (ganglioneuromas, ganglioneuroblastomas and neuroblastomas) and correlate them with different clinical parameters (age, sex, diagnosis, Shimada index, proliferation index, tumor location, and 1p and v-myc avian myelocitomatosis viral-related (MYCN) status) in order to find new molecular and/or prognostic markers for neuroblastoma. To assess the genomic damage in neuroblastic tumors, we used an arbitrarily primed PCR approach, a technique based on the reproducibility of band profiles obtained by a PCR with a low annealing temperature in its first cycles. Genomic damage was assessed by comparing band profiles of tumors and normal paired samples. Gains and losses in the intensity of the bands were computerized and referred to the total number of bands analyzed. We found a higher genomic damage fraction (GDF) in the female's group (U-Mann-Whitney, P = 0.025), but we could not find any association between GDF and tumor location, proliferation index, diagnosis or age of the patient. There was no relationship between 1p status and GDF, but tumors with MYCN amplification had a slightly higher GDF. MYCN amplification might in some way contribute to genomic instability of neuroblastomas.

Erratum of “The Clinical Consequences of Massive Genomic Imbalances in Human Cancers” doi.org/10.1155/2005/430568

Abstract Introduction: While somatic mutations in colorectal cancer (CRC) are well characterized, little is known about the accumulation of cancer-associated mutations in normal colon. This knowledge is crucial to understand the origin and evolution of cancer and to improve cancer prevention and prediction. The study of somatic mutation within normal tissue, however, is technically challenging because these mutations exist at extremely low frequency limiting their detection by conventional next-generation sequencing (NGS). Here we used Duplex Sequencing (DS), an error correction NGS approach that enables extreme sensitivity for mutation detection, to characterize the mutational landscape of cancer-associated genes in normal colonic mucosa of patients with and without CRC. Methods: We used a DS-based method called CRISPR-DS, which employs double stranded molecular tagging for ultra-accurate sequencing and CRISPR-Cas9 target enrichment for efficient library conversion. A panel including the full coding region of TP53 and the hotspot codons of BRAF, KRAS and PIK3CA was designed to sequence at high depth (mean 2,500x) the DNA from normal colon mucosa of 47 patients, 23 of them with CRC cancer (mean age 57 years) and 24 without (mean age 59 years). Normal colon tissue from CRC patients was obtained distant from the tumor (>15cm). Mutation frequency was calculated for all target genes and for TP53 only as the number of mutated reads (overall and separately for coding and non-coding regions) divided by the total nucleotides sequenced. Results: 168 mutations (124 coding and 44 non-coding) were identified. In nearly all patients (43/47), normal colon carried at least one coding mutation in a cancer associated gene included in the panel. Compared to patients without cancer, the normal colon of patients with CRC showed higher mutation frequency overall (5.5x10-7 vs 3.6x10-7, p=0.009) and in coding regions (7.5x10-7 vs 4.2x10-7, p=0.003), but not in non-coding regions. Similar results were observed for overall TP53 mutations (6.4x10-7 vs 3.7x10-7, p=0.01) and TP53 coding mutations (9.5x10-7 vs 4.9x10-7, p=0.006). Both groups of patients carried cancer-associated TP53 mutations but in patients with CRC the proportion of common CRC mutations and pathogenic mutations was significantly higher (p=0.01 and p=0.02, respectively). KRAS hotspot mutations (codons 12 and 13) were identified in the normal colon of 30% (7/23) of patients with CRC but in none of the patients without CRC (0/24). TP53 hotspot mutations were observed in 22% (5/23) patients with CRC and in 13% (3/24) of patients without cancer. Overall, 44% (10/23) of patients with CRC carried mutations in hotspots vs 25% (6/24) of patients without cancer and half of the CRC patients with hotspot mutations carried hotspot mutations in more than one gene. Conclusions: Common CRC mutations are present in the normal colon of adult individuals but at higher frequency and with more pathogenicity in those that have developed CRC. These results indicate that somatic evolution takes place in the normal human colon but this process appears to be increased in patients with CRC. Because the biopsies analyzed were collected more than 15cm away from tumors, these results raise questions about the extension of precarcinogenic fields harboring clonal expansions and prompts to further investigation into the landscape of cancer-associated mutations in non-cancerous colonic mucosa. Citation Format: Júlia Matas Gironella, Brendan Kohrn, Jeanne Fredickson, Kelly Carter, Ting Wang, Ming Yu, William M Grady, Miguel Angel Peinado, Rosa Ana Risques. Somatic evolution in normal colon of patients with and without cancer [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2021; 2021 Apr 10-15 and May 17-21. Philadelphia (PA): AACR; Cancer Res 2021;81(13_Suppl):Abstract nr LB231.