Figure 4

<p>LINE-1 methylation variability in cancer cell lines. DNA samples of normal peripheral blood lymphocyte, normal colon mucosa and sixty-one cell lines from eight different tissues types were investigated for LINE-1 methylation using bisulfite PCR followed by pyrosequencing. The normal tissues presented high levels of LINE-1 methylation (above 70% in average), and a large variation in methylation levels was observed for cancer cell lines, with a minimum methylation density of 6.5% being observed for the leukemia cell line K562. Taken as a group, leukemia cell lines were moderately demethylated (average 56.1%), followed by ovary, colon, prostate and lung cancer cell lines (variation from 49.7% to 35.1%). Central nervous system (CNS), breast and the one liver cancer cell lines tested were deeply demethylated (bellow 30% in average). Dotted line represents average methylation in normal controls.</p

Figure 1

<p>Quantitation of DNA methylation using bisulfite LINE-1 PCR and pyrosequencing. A) Diagram of the CpG island promoter (GenBank accession no. X58075, nucleotide position 108–520 bp) associated with the full length LINE-1. Each vertical line represents a single CpG site. The 3′UTR, 5′UTR and two ORFs of LINE-1 are shown at the top. Arrows indicate the location of primers used for bisulfite PCR (R-biot and F) and pyrosequencing (S). B) Representative LINE-1 pyrograms for normal peripheral blood lymphocytes (PBL) and breast cancer cell lines (MB-468 and SKBR3). The pyrogram quantitates C for methylated and T for unmethylated DNA. The shaded region represents the CpG site quantitated in LINE-1 elements, and the percent methylation is shown above the peak.</p

Figure 3

<p>Methylated CpG Island Amplication (MCA)/CpG island microarray for repetitive DNA sequences. A) Relative abundance of hypermethylated and hypomethylated repeats for each CIMP/MSI group. A higher number of hypermethylated compared to hypomethylated repeats was observed for the CIMP+/MSI+group, and a gradual change in representation of hypermethylated and hypomethylated repeats was seen for the CIMP+/MSI-and CIMP-/MSI-groups, resulting in an overrepresentation of hypomethylated repeats in microsatellite stable groups. B) Validation of microarray results for LINE repeats. Note that CIMP/MSI groups with higher demethylation, as determined by bisulfite-pyrosequencing of LINE-1, presented also a higher number of hypomethylated LINE repeats by microarray analysis, as represented by a lower hyper/hypomethylation ratio. C) Unsupervised hierarchical clustering was applied to methylation data from a set of 770 repetitive DNA sequences across sixteen colorectal tumors paired with their normal appearing mucosa DNA. The colorectal tumors dendrogram is shown, and the sample ID for each case is included in the right. The terminal branches are color coded to represent the CIMP/MSI status of the tumor sample (red, CIMP+/MSI+; blue, CIMP+/MSI-; green, CIMP-/MSI-). Overall, samples of the same CIMP/MSI group clustered together, reinforcing the different methylation fate for repetitive DNA sequences methylation in each group. LINE, long interspersed nuclear elements; SINE, short interspersed nuclear elements, LTR, long terminal repeats; DNA repeats; Satellite repeats.</p

Figure 2

<p>Differential LINE-1 methylation among CIMP/MSI groups in primary colorectal carcinoma samples (CRCs). A) Colorectal tumor DNA and their normal appearing adjacent mucosa from sixty patients were evaluated for LINE-1 methylation. These tumors were previously evaluated for CpG island methylator phenotype (CIMP), using a panel of single-copy genes methylation analysis, and microsatellite instability (MSI) status, resulting in the identification of three CIMP/MSI groups. In normal appearing mucosa (top) little variation in LINE-1 methylation is observed between samples and CIMP/MSI groups (average methylation = 64.3%), while in tumor (bottom) several samples undergo high LINE-1 demethylation (25/60 tumor samples have methylation density bellow 55%), most notable in CIMP+/MSI-and CIMP-/MSI-groups. B) Relative LINE-1 demethylation in CRCs. Relative demethylation was calculated as the percent change of LINE-1 methylation in tumor compared to its normal appearing mucosa. Both CIMP+/MSI-and CIMP-/MSI-samples presented in average 16% demethylation for LINE-1, while no significant changes were observed for the CIMP+/MSI+samples. For the CIMP+group, 4–9% increase of methylation density for LINE-1 was observed for a small fraction of samples, most of them identified as CIMP+/MSI+samples.</p

Methylation density of LINE-1and clinical and demographic characteristics of 60 colorectal carcinomas and their normal appearing adjacent mucosa^*

*<p>Means of cancer methylation is significantly (P<0.001) lower than mean of adjacent normal for all categories except for MSI+cancers (p = 0.24)</p>a<p>Significant P values (<0.05) are underlined</p>b<p>Side information was not available for all cases</p><p>CI = confidence interval</p

Genomic analysis of head and neck cancer cases from two high incidence regions

<div><p>We investigated how somatic changes in HNSCC interact with environmental and host risk factors and whether they influence the risk of HNSCC occurrence and outcome. 180-paired samples diagnosed as HNSCC in two high incidence regions of Europe and South America underwent targeted sequencing (14 genes) and evaluation of copy number alterations (SCNAs). <i>TP53</i>, <i>PIK3CA</i>, <i>NOTCH1</i>, <i>TP63</i> and <i>CDKN2A</i> were the most frequently mutated genes. Cases were characterized by a low copy number burden with recurrent focal amplification in 11q13.3 and deletion in 15q22. Cases with low SCNAs showed an improved overall survival. We found significant correlations with decreased overall survival between focal amplified regions 4p16, 10q22 and 22q11, and losses in 12p12, 15q14 and 15q22. The mutational landscape in our cases showed an association to both environmental exposures and clinical characteristics. We confirmed that somatic copy number alterations are an important predictor of HNSCC overall survival.</p></div

Workflow of processing and analysis of HNSCC samples from the three different studies.

<p>QC for copy number evaluation: Quality control of samples based on signal to noise ratio>5.0. Maps show estimated age-standardized incidence rates for HNSCC (other pharynx sites) in Europe and South America. [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref031" target="_blank">31</a>].</p

OncoPrint diagram of mutational frequencies and types of alterations of the 14 genes sequenced.

<p>Only altered samples are shown. Rows are sorted based on the frequency of the alterations in all samples and columns are sorted to visualize the mutual exclusivity across genes. Frequency of mutations for the following Head and Neck cancer publications are shown: Head & Neck (TCGA)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref010" target="_blank">10</a>], Head & Neck (JHU)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref039" target="_blank">39</a>], Head & Neck (Broad)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref032" target="_blank">32</a>], Head & Neck (MDA)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref040" target="_blank">40</a>], Head & Neck (MSKCC)[<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191701#pone.0191701.ref041" target="_blank">41</a>]. NA: Not available.</p

Kaplan-Meier curves showing overall survival outcome for nodal status, significant focal copy number alterations in 22q11.2,15q22 and 12p12 regions associated to smoking and advanced stage, amplification in 4p16.3 and for the three SCNAs clusters.

<p>Kaplan-Meier curves showing overall survival outcome for nodal status, significant focal copy number alterations in 22q11.2,15q22 and 12p12 regions associated to smoking and advanced stage, amplification in 4p16.3 and for the three SCNAs clusters.</p

Integrative cluster analysis plot.

<p>Cases are grouped by mutation and SCNA status. Top panel: only significant clustering genes are shown (0 = non-mutated, 1 = mutated), middle panel: SCNAs. Amplified (red) and deleted (blue) chromosomal regions. Altered regions are arranged vertically and sorted by genomic locus, with chromosome 1 at the top of the panel and chromosome 22 at the bottom, lower panel: colour coded clinical and epidemiological characteristics.</p